Research ArticlePhysicochemical and Biological Characterization ofa Biosimilar Trastuzumab
Carlos A Loacutepez-Morales1 Mariana P Miranda-Hernaacutendez1
L Carmina Juaacuterez-Bayardo1 Nancy D Ramiacuterez-Ibaacutentildeez1 Alexis J Romero-Diacuteaz1
Nelly Pintildea-Lara1 Viacutector R Campos-Garciacutea2 Neacutestor O Peacuterez1
Luis F Flores-Ortiz1 and Emilio Medina-Rivero1
1Unidad de Investigacion y Desarrollo Probiomed SA de CV Cruce de Carreteras Acatzingo-Zumpahuacan sn52400 Tenancingo MEX Mexico2MUHC Research Institute Royal Victoria Hospital McGill University 687 Pine Avenue West Montreal QC Canada H3A 1A1
Correspondence should be addressed to Luis F Flores-Ortiz luisfloresprobiomedcommx andEmilio Medina-Rivero emiliomedinaprobiomedcommx
Received 11 October 2014 Revised 10 March 2015 Accepted 24 March 2015
Academic Editor Pedro H Oliveira
Copyright copy 2015 Carlos A Lopez-Morales et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
According to the World Health Organization the incidence of malignant neoplasms and endocrine blood and immune disorderswill increase in the upcoming decades along with the demand of affordable treatments In response to this need the development ofbiosimilar drugs is increasingworldwideThe approval of biosimilars relies on the compliancewith international guidelines startingwith the demonstration of similarity in their physicochemical and functional properties against the reference product Subsequentclinical studies are performed to demonstrate similar pharmacological behavior and to diminish the uncertainty related to theirsafety and efficacy Herein we present a comparability exercise between a biosimilar trastuzumab and its reference product by usinga hierarchical strategy with an orthogonal approach to assess the physicochemical and biological attributes with potential impacton its pharmacokinetics pharmacodynamics and immunogenicity Our results showed that the high degree of similarity in thephysicochemical attributes of the biosimilar trastuzumab with respect to the reference product resulted in comparable biologicalactivity demonstrating that a controlled process is able to provide consistently the expected product These results also constitutethe basis for the design of subsequent delimited pharmacological studies as they diminish the uncertainty of exhibiting differentprofiles
1 Introduction
Biopharmaceutical products containing chimeric human-ized or fully human monoclonal antibodies (mAbs) areamong the most successful and demanded therapies due totheir highly specific mechanisms of action that result in animprovement of the patientsrsquo conditions and an increase inthe survival rate while minimizing the adverse side-effectswhen compared to other treatments [1] Consequently newmanufacturing sites process scale-ups as well as processimprovements contribute to the well-known heterogeneitynaturally present in biotherapeutic products For this pur-pose the ICH Q5 E guideline provides the principles for
assessing comparability of licensed biotechnological productssubject to process changes throughout their life cycle [2]
In this sense the approval of biosimilar products whichhave been recognized not only as an alternative but as anecessity to increase health coverage and improve the qualityof life of patients follows a similar comparability schemeInternational guidelines on biosimilarity [3ndash5] outline thatthe approval of biosimilars must rely on the demonstration ofcomparability towards the reference product starting with anexhaustive physicochemical and biological characterizationwhose results will provide evidence to support the extent ofadditional clinical evaluation [6ndash8]
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 427235 10 pageshttpdxdoiorg1011552015427235
2 BioMed Research International
For this purpose the proper identification of criticalquality attributes (CQAs) that may impact on the phar-macokinetics pharmacodynamics and immunogenicity canbe achieved through a deep knowledge of the chemicalcomposition and the higher order structure of the activepharmaceutical ingredient (API) contained in the referenceproduct as well as the known relationships between specificattributes and biological functionality anticipated by thebiotechnological industry and the scientific community [9ndash17] Furthermore the ICHQ9guideline highlights the need ofevaluating the quality of a biopharmaceutical product basedon a risk analysis that considers relevant attributes to thedrugrsquos safety and efficacy [18]
In this work we present a comparability study betweena biosimilar trastuzumab and its reference product Trastuz-umab is a humanized monoclonal antibody targeted againstthe extracellular portion of the human epidermal growthfactor receptor (HER2 p185) which is overexpressed inapproximately 15 to 30 of the invasive breast cancer cases[19ndash22] The chemical physical and functional propertiesclosely related to its pharmacological behaviorwere identifiedthrough a risk analysis then those CQAs were evaluatedusing several analytical techniques in an orthogonal approachthat increases the reliability of the results obtained
2 Materials and Methods
21 Materials Biosimilar trastuzumab (440mg powder forconcentrate for solution for infusion) from Probiomed SAdeCV (MexicoCityMexico) andHerceptin (440mgpowderfor concentrate for solution for infusion) from F HoffmannLa Roche Ltd (Basel Switzerland) were used for the compa-rability study
22 Methods
221 Physicochemical Properties Primary sequences ver-ified from the whole-molecule exact masses and trypticpeptide mappings were analyzed by reverse phase ultra-performance-liquid-chromatography coupled to a tandemquadrupoletime-of-flight mass spectrometer (RP-UPLC-MSMS) Higher order structure was evaluated by differen-tial scanning calorimetry (DSC) circular dichroism (CD)and fluorescence lifetime using the time correlated singlephoton counting technique (TCSPC) Charge heterogene-ity of the whole carboxypeptidase-digested and papain-digested molecule was assessed either by capillary isoelec-trofocusing (cIEF) or by cation exchange ultra-performance-liquid-chromatography (CEX-UPLC) Purity was deter-mined by capillary gel electrophoresis under reducing (CGE-R) and nonreducing (CGE-NR) conditions and size exclu-sion ultra-performance-liquid-chromatography (SE-UPLC)Sample treatment and analysis conditions were performedas previously described for RP-UPLC-MSMS DSC CDCEX CGE-R and CGE-NR by Flores-Ortiz et al 2014 [23]TCSPC by Perez Medina Martınez et al 2014 [24] cIEF byEspinosa-de la Garza et al [25]
N-linked glycans were released from trastuzumabby enzymatic hydrolysis using PNGase F from New Eng-land Biolabs Inc (IpswichMA) and thenwere labeledwith 8-aminopyrene-136-trisulfonic acid (APTS) and analyzed bycapillary zone electrophoresis (CZE) [26]The electrophoret-ic separationwas carried out in a PA 800 plus Analysis SystemfromBeckmanCoulter Inc (Brea CA) using an amine coatedcapillary of 50 120583m ID times 502 cm total length with 40 cmeffective length at 20∘C Laser induced fluorescence (LIF)detection was used at an excitation wavelength of 488 nmand emission band-pass filter of 520 nm An orthogonalanalysis was performed by hydrophilic interaction ultra-performance-liquid-chromatography (HILI-UPLC) afterlabeling with 2-aminobenzoic acid (2-AB) following apreviously reported methodology [27]
23 Functional Properties
231 Fc120574RIIIa Affinity by Isothermal Titration Calorime-try (ITC) Affinity constants under equilibrium (119870
119886) were
obtained from a Nano ITC instrument (TA Instruments IncNew Castle DE) 300 120583L of Fc120574RIIIa solutions at 50 120583Min PBS at pH 72 was titrated with continuous injectionsof 19 120583L trastuzumab solutions at 50 120583M in PBS at pH72 until saturation at 25∘C NanoAnalyze Software v241(TA Instruments Inc New Castle DE) was used for theintegration of heat signals and nonlinear regression analysisof the data
232 FcRn Affinity by BLI Binding kinetics of trastuzumabto FcRn were determined using a Bio-Layer Interferometry(BLI) instrument Octet QK384 from Pall ForteBio Corp(Menlo Park California) Biotinylated FcRnwas immobilizedto biosensors coated with streptavidin Binding profiles weredisplayed by sensograms Global kinetic analyses were deter-mined using a 2 1 heterogeneous ligand model fit using R-linked analysis
233 HER2 Affinity Assay HER2 expressing cells SK-BR-3(ATCC HTB-30) were incubated in the presence of differentconcentrations of trastuzumab in McCoy-5A medium with10 FBS for 2 h at 37∘C HRP-conjugated goat anti-humanIgG was added to detect the trastuzumabndashSK-BR-3 complexafter 1 h of incubation at 37∘C using TMB as substrate for30min at room temperature Absorption was measured at450 nm Test results were expressed as the relative percentageof the EC
50from the dose-response curve of the biosimilar
trastuzumab with respect to the reference product
234 Antiproliferation Assay BT-474 cells (ATCC HTB-20)were seeded in DMEMmedia with 10 FBS 1 nonessentialamino acids and incubated at 37∘C Different concentrationsof trastuzumab were added with further incubation for 8days Crystal violet was added to stain the cells for 15minat room temperature followed by fixation with formaldehydeand water rising Acetic acid aqueous solution (33 vv) wasadded to remove the dye excess absorbance was measured at540 nm Test results were expressed as the relative percentage
BioMed Research International 3
Physicochemical characterization
Physical characterization
Functional assays
Comparability
Charge- CEX CZE HIC cIEF
Glycosylation- HILIC CZE
Degradation- CGE-NR
Purity- CGE-R SEC AUC CZE
Isoelectric point- cIEF
Identity- MS peptide mapping
SDS-PAGE WB
Size- SEC
Massradii- SEC-MALS MS
- FL TCSPCDSC CD HDX Ellman
Aggregation- ESZ SEC AUC
Biological activity- ADCC CDC
antiproliferation assayAffinity
- ITC BLI SPRELISA
Structure
Figure 1 Characterization strategy performed for Trastuzumab-Probiomed
Figure 2 Mirror plot of peptide mapping chromatograms obtained from RP-UPLC-UV for Trastuzumab-Probiomed (upper) and thereference product (lower)
of the EC50
from the dose-response curve of the biosimilartrastuzumab with respect to the reference product
3 Results and Discussion
Our characterization strategy (Figure 1) comprised a set ofstate-of-the-art analytical techniques planned for a hierarchi-cal study of a biosimilar trastuzumab using an orthogonalapproach CQAs were identified using a risk analysis consid-ering each of the physicochemical and functional propertiesthat may have an impact on efficacy (pharmacokineticsand pharmacodynamics) and safety (immunogenicity) oftrastuzumab (Table 1) [9ndash17] In this work only certainmethodologies were selected to depict a global overview
of the characterization study Hereafter CQAs were classi-fied by their physicochemical physical or biological natureand analyzed comparatively for a biosimilar trastuzumab(Trastuzumab-Probiomed) and its reference product
31 Physicochemical Properties The identity of Trastuzumab-Probiomed towards the reference product was determinedby the correspondence of their tryptic peptide mappings(Figure 2) MSMS analysis verified the amino acid sequenceof both products against the theoretical stated on the inven-tion patent of trastuzumab [28] unveiling a sequence match-ing of 998 and 993 for the heavy chain and 995 and995 for the light chain for both Trastuzumab-Probiomedand the reference product respectively (Figures 3 and 4)
Figure 3 Sequence coverage of the heavy and light chains of Trastuzumab-Probiomed obtained from the MSMS analysis
This correspondence was further confirmed by the analysesof the exact masses against the theoretical mass [28 29]for both whole and deglycosylated molecules (Tables 2 and3) The sequences coverage confirms that the amino acidsequence of Trastuzumab-Probiomed is identical to thereference product while the lt25Da observed differences inintact masses for the whole molecule below the expectedwidth of the isotopic pattern distribution of a mAb showin advance a comparable degree of heterogeneity due toposttranslational modifications in both products ultimatelyproducing an equivalent immunogenic response
Regarding glycan microheterogeneity which is known tocontribute to the correct folding and stability of a mAb itwas analyzed by CZE and HILI-UPLC Particularly highlymannosylated and sialylated glycoforms are reported toalter a mAb half-life in blood and are linked to potentialimmunogenic responses moreover effector functions can bealtered due to the presence of highly mannosylated bisectedand fucosylated glycoforms as a consequence of charge orsteric hindrances [10ndash12]
CZE analyses revealed that the glycan patterns ofTrastuzumab-Probiomed and the reference product arecomprised of the same principal glycoforms (Figure 5(a))showing amean relative abundance of galactosylated variantsof 6601 and 4957 plusmn 618 (CI 95) for Trastuzumab-Probiomed and the reference product respectively which isnot expected to have an impact on the functional propertiessince galactosylation has not been reported to alter themechanisms of action of mAbs as confirmed by the affinitiesand biological potency analyses discussed below Further
analysis by HILI-UPLC of the glycoforms identified ascritical for PK PD or immunogenicity (Table 1) revealedcomparable relative abundances of highly mannosylatedvariants being 200 plusmn 010 (CI 95) and 396 plusmn 045 (CI 95)for Trastuzumab-Probiomed and the reference productrespectively whereas the mean abundance for hybrid andsialylated variants was 475 plusmn 019 (CI 95) and 027 plusmn008 (CI 95) for the reference product and 295 plusmn 015 (CI95) and 106 plusmn 014 (CI 95) for Trastuzumab-Probiomedrespectively These results confirm similarity of the criticalglycoforms between Trastuzumab-Probiomed and thereference product thus similar PK and PD profiles and nodifferential immunogenicity response are expected
On the other hand charge heterogeneity evaluatedthrough cIEF analysis revealed that isoelectric points (pI) forthemain isoformwere 869plusmn 000 (CI 95) for Trastuzumab-Probiomed and 870 plusmn 001 (CI 95) for the referenceproduct in accordancewith the expected pI variations duringmanufacturing no larger than 02 units [15 16]The observedisoform-abundance-weighted pI values confirmed similarityof charge heterogeneity among products being 860 plusmn 001(CI 95) for Trastuzumab-Probiomed and 861 plusmn 001 (CI95) for the reference product It has been reported that onlychanges in one pI unit can significantly alter the therapeuticactivity of a mAb thus the observed variation is not expectedto affect the clinical behavior of Trastuzumab-Probiomedwith respect to the reference product
An orthogonal analytical technique for the evaluationof charge heterogeneity was CEX-UPLC which revealedthat the averaged abundances of the main acidic and basic
6 BioMed Research International
S C A A S G F N I K
S A D T S K N T A Y
A S T K G P S V F P
H T F P A V L Q S S
K S C D K T H T C P
H E D P E V K F N W
E Y K C K V S N K A
L V K G F Y P S D I
Q Q G N V F S C S V
I T C R A S Q D V N
F T L T I S S L Q P
S D E Q L K S G T A
S T Y S L S S T L T
D T Y I H W V R Q A
L Q M N S L R A E D
L A P S S K S T S G
P C P A P E L L G G
G L Y S L S S V V T
Y V D G V E V H N A
L P A P I E K T I S
A V E W E S N G Q P
M H E A L H N H Y T
T A V A W Y Q Q K P
E D F A T Y Y C Q Q
S V V C L L N N F Y
L S K A D Y E K H K
A RK
P G K G L E W V A R
T A V Y Y C S R W G
G T A A L G C L V K
V P S S S L G T Q T
P S V F L F P P K P
K T K P R E E Q Y N
K G Q P E P Q
E N N Y K T T P P V
Q K S L S L S P G K
G K A P K L L I Y S
H Y T T P P T F G Q
P R E A K V Q W K V
VY A C E V T H Q G
E V Q L V E S G G G
I Y P T N G Y T R Y
G D G F Y A M D Y W
D Y F P E P V T V S
Y I C N V N H K P S
K D T L M I S R T P
S T Y R V V S V L T
V Y T L P P S R E E
L D S D G S F F L Y
R
D I Q M T Q S P S S
A S F L Y S G V P S
G T K V E I K T V
D N A L Q S G N S Q
L S S P V T K S F N
L V Q P G G S L R L
A D S V K G R F T I
G Q G T L V T V S S
W N S G A L T S G V
N T K V D K K V E P
E V T C V V V D V S
V L H Q D W L N G K
M T K N Q V S L T C
S K L T V D K S R W
L S A S V G D R V T
R F S G S R S G T D
A A P S V F I F P P
E S V T E Q D S K D
RG E C
Control coverage () 995 Control unique coverage () 995
Figure 4 Sequence coverage of the heavy and light chains of the reference product obtained from the MSMS analysis
EU
G0F G1F
G1F998400
G2F
(min)
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
(a)
900
800
700
600
500
400
300
200
100
060 65 70 75 80 85 90
Temperature (∘C)
Fit (120583
J)
(b)
Figure 5 (a) Glycan profile for the reference product (upper) and Trastuzumab-Probiomed (lower) (b) Thermostability by DSC for thereference product (lower) and Trastuzumab-Probiomed (upper)
isoforms were within the same order of magnitude for bothproducts being the mean values of 570 332 and 98(119899 = 3) for Trastuzumab-Probiomed and 625 273and 103 (119899 = 3) for the reference product respectivelyFurthermore the results obtained after digestion with car-boxypeptidase B showed also a comparable content of basic
acidic and main isoforms among the two products with amain relative content of 164 306 and 530 (119899 = 3) forthe reference product and 88 378 and 534 (119899 = 3) forTrastuzumab-Probiomed respectively
After papain digestion the mean abundance of basicisoforms in the reference product (119899 = 3) was 38 for
the Fc fragment and 49 for the Fab fragment whereasfor Trastuzumab-Probiomed (119899 = 3) it was 42 for theFc fragment and 65 for the Fab fragment Regardingacidic isoforms the mean abundance was 33 for the Fcfragment and 167 for the Fab fragment of the referenceproduct while for Trastuzumab-Probiomed it was 37 forthe Fc fragment and 161 for the Fab fragment Finally theabundance of the Fc and Fab fragments was 267 and 446respectively for the reference product and for Trastuzumab-Probiomed the abundance of the Fc and Fab fragments was257 and 438 respectively
Overall the results from cIEF and CEX-UPLC show thatboth products exhibit comparable charge heterogeneitieseither as a whole molecule or as the fragments responsible forthe recognition and effector functions of trastuzumab thusno differences in functional activity should be expected
CGE-NR and SE-UPLC results demonstrated that bothproducts have a similar degree of purity (Tables 4 and 5)based on the relative content of monomer with respect tothe presence of aggregates and other degraded or truncatedisoforms It is known that protein aggregation can induceimmunogenicity although a small amount of aggregates isexpected this amount is likely to increase due to stressconditions that a mAb may undergo during its manufacturepurification formulation and shelf-life [9 30] Aggregationmay reveal new epitopes that potentially could stimulate theproduction of anti-drug antibodies (ADAs) resulting in theloss of activity immunogenic reactions or adverse effectsduring administration Likewise the presence of fragmentsor truncated forms coming from hydrolysis reactions couldnegatively impact on the safety and therapeutic effect of amAb [31 32] The content of aggregates and truncated forms
Table 4 Monomer content of trastuzumab by SE-UPLC and CGE-NR Variation is presented as confidence intervals at 95 (119899 = 3)
of Trastuzumab-Probiomed were lower than the limits estab-lished by the USP [29] and were comparable to the referenceproduct thus the risk of developing a different immunogenicresponse (differential immunogenicity) is diminished
32 Physical Properties Since the functionality of trastuz-umab is affected by its three-dimensional structure whichresults from its primary sequence and posttranslationalmod-ifications that alter its size mass folding and stability [8]we performed analyses to assess the spatial configuration ofTrastuzumab-Probiomed compared to its reference productTime correlated single photon counting analysis (TCSPC)was employed to evaluate the fluorescence lifetime (120591) whichdepends on the exposure of aromatic amino acids within theprotein thus demonstrating similarity when the results areobtained from comparative analyses [33ndash36] TCSPC resultsshowed that the averaged 120591 of Trastuzumab-Probiomed was343Eminus09 plusmn 139Eminus10 s (CI 95) while the averaged 120591 forthe reference product was 349Eminus09 plusmn 169Eminus11 s (CI 95)Regarding CD the obtained spectrograms were superimpos-able in both near- and far-UV regions (Figure 6) suggestingthat alpha helix beta sheets random coil disulfide bondsand aromatic amino acids are distributed in a comparablespatial arrangement Finally transition temperatures (119879
119898) of
Trastuzumab-Probiomed (119899 = 3) by DSC (Figure 5(b)) were704∘C 791∘C 810∘C and 825∘C whereas for the referenceproduct (119899 = 3) they were 705∘C 796∘C 812∘C and 827∘Cfor both products the CI at 95 was lt002∘C for all thetemperatures Collectively TCSPC CD andDSC determinedthat thermostability and secondary and tertiary structures ofTrastuzumab-Probiomed were comparable to the referenceproduct In particular thermostability results are indicative ofa proper protein folding of both products in their respective
8 BioMed Research International
Table 5 Relative abundance of trastuzumab subunits by CGE-R Variation is presented as confidence interval at 95 (119899 = 3)
HC heavy chain NGHC nonglycosylated heavy chain and LC light chain
7
0
minus10
minus17240 250 300 350
Wavelength (nm)
CD (m
deg
)
(a)
30
20
10
0
minus10
minus20200 220 240 260 280 300
Wavelength (nm)CD
(m d
eg)
(b)
Figure 6Analysis of the three-dimensional structure of trastuzumabbyCDofTrastuzumab-Probiomed (solid line) and the reference product(dotted line) in both near-UV region (a) and far-UV region (b)
3
2
1
1
0
10 100 1000 10000
OD
(450
nm)
Trastuzumab (ngmL)
(a)
10 100 1000 10000
Trastuzumab (ngmL)
285
235
185
135
085
035
OD
(540
nm)
(b)
Figure 7 Comparison of in vitro activity between Trastuzumab-Probiomed and the reference product (a) Curve of binding affinity to HER2(b) potency curve obtained from the antiproliferation assay the solid line corresponds to Trastuzumab-Probiomed while the dashed linecorresponds to the reference product
formulation This physicochemical and physical similarity isthe major contributor to equivalent biological and functionalresponses
33 Functional Properties The relative affinity of Trastuzum-ab-Probiomed towards its targetmoleculeHER2 (Figure 7(a)and Table 7) was evaluated with respect to the referenceproduct resulting in an averaged relative affinity of 977Thus it is expected that Trastuzumab-Probiomed can exert itsactivity through the reported mechanisms of action includ-ing HER2 downregulation prevention of the heterodimerformation initiation of Gl arrest induction of p27 andprevention of HER2 cleavage [37]
The main mechanisms of action rely on the affinityof the Fc fragment of trastuzumab towards Fc120574 receptorsFor instance Fc120574RIIIa present on effector cells such asmacrophages monocytes and natural killer cells activatesand induces ADCC mechanism against HER2-positive cells[37 38] Binding affinities towards Fc120574RIIIa were evalu-ated by ITC being the averaged affinity constants (119870
119886) of
261 plusmn 054E+06Mminus1 for Trastuzumab-Probiomed and 248 plusmn030E+06Mminus1 for the reference product (Table 6) Likewisethe mean dissociation constants (119870
119863) to FcRn which regu-
lates IgG catabolism were determined by BLI as 258Eminus07Mplusmn 102Eminus07M (CI 95) for Trastuzumab-Probiomed with arelative binding affinity of 1143 (119899 = 3) with respect to
BioMed Research International 9
Table 6 Affinity of trastuzumab to Fc120574RIIIa
Product Batch Affinity constant (119870119886
) toFc120574RIIIa (Mminus1)
Trastuzumab-Probiomed
TZPP14001 271119864 + 06
TZPP12002 286119864 + 06
TZPP12003 225119864 + 06
Reference productN35893 266119864 + 06
N35812 248119864 + 06
N36003 231119864 + 06
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
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[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
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[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
For this purpose the proper identification of criticalquality attributes (CQAs) that may impact on the phar-macokinetics pharmacodynamics and immunogenicity canbe achieved through a deep knowledge of the chemicalcomposition and the higher order structure of the activepharmaceutical ingredient (API) contained in the referenceproduct as well as the known relationships between specificattributes and biological functionality anticipated by thebiotechnological industry and the scientific community [9ndash17] Furthermore the ICHQ9guideline highlights the need ofevaluating the quality of a biopharmaceutical product basedon a risk analysis that considers relevant attributes to thedrugrsquos safety and efficacy [18]
In this work we present a comparability study betweena biosimilar trastuzumab and its reference product Trastuz-umab is a humanized monoclonal antibody targeted againstthe extracellular portion of the human epidermal growthfactor receptor (HER2 p185) which is overexpressed inapproximately 15 to 30 of the invasive breast cancer cases[19ndash22] The chemical physical and functional propertiesclosely related to its pharmacological behaviorwere identifiedthrough a risk analysis then those CQAs were evaluatedusing several analytical techniques in an orthogonal approachthat increases the reliability of the results obtained
2 Materials and Methods
21 Materials Biosimilar trastuzumab (440mg powder forconcentrate for solution for infusion) from Probiomed SAdeCV (MexicoCityMexico) andHerceptin (440mgpowderfor concentrate for solution for infusion) from F HoffmannLa Roche Ltd (Basel Switzerland) were used for the compa-rability study
22 Methods
221 Physicochemical Properties Primary sequences ver-ified from the whole-molecule exact masses and trypticpeptide mappings were analyzed by reverse phase ultra-performance-liquid-chromatography coupled to a tandemquadrupoletime-of-flight mass spectrometer (RP-UPLC-MSMS) Higher order structure was evaluated by differen-tial scanning calorimetry (DSC) circular dichroism (CD)and fluorescence lifetime using the time correlated singlephoton counting technique (TCSPC) Charge heterogene-ity of the whole carboxypeptidase-digested and papain-digested molecule was assessed either by capillary isoelec-trofocusing (cIEF) or by cation exchange ultra-performance-liquid-chromatography (CEX-UPLC) Purity was deter-mined by capillary gel electrophoresis under reducing (CGE-R) and nonreducing (CGE-NR) conditions and size exclu-sion ultra-performance-liquid-chromatography (SE-UPLC)Sample treatment and analysis conditions were performedas previously described for RP-UPLC-MSMS DSC CDCEX CGE-R and CGE-NR by Flores-Ortiz et al 2014 [23]TCSPC by Perez Medina Martınez et al 2014 [24] cIEF byEspinosa-de la Garza et al [25]
N-linked glycans were released from trastuzumabby enzymatic hydrolysis using PNGase F from New Eng-land Biolabs Inc (IpswichMA) and thenwere labeledwith 8-aminopyrene-136-trisulfonic acid (APTS) and analyzed bycapillary zone electrophoresis (CZE) [26]The electrophoret-ic separationwas carried out in a PA 800 plus Analysis SystemfromBeckmanCoulter Inc (Brea CA) using an amine coatedcapillary of 50 120583m ID times 502 cm total length with 40 cmeffective length at 20∘C Laser induced fluorescence (LIF)detection was used at an excitation wavelength of 488 nmand emission band-pass filter of 520 nm An orthogonalanalysis was performed by hydrophilic interaction ultra-performance-liquid-chromatography (HILI-UPLC) afterlabeling with 2-aminobenzoic acid (2-AB) following apreviously reported methodology [27]
23 Functional Properties
231 Fc120574RIIIa Affinity by Isothermal Titration Calorime-try (ITC) Affinity constants under equilibrium (119870
119886) were
obtained from a Nano ITC instrument (TA Instruments IncNew Castle DE) 300 120583L of Fc120574RIIIa solutions at 50 120583Min PBS at pH 72 was titrated with continuous injectionsof 19 120583L trastuzumab solutions at 50 120583M in PBS at pH72 until saturation at 25∘C NanoAnalyze Software v241(TA Instruments Inc New Castle DE) was used for theintegration of heat signals and nonlinear regression analysisof the data
232 FcRn Affinity by BLI Binding kinetics of trastuzumabto FcRn were determined using a Bio-Layer Interferometry(BLI) instrument Octet QK384 from Pall ForteBio Corp(Menlo Park California) Biotinylated FcRnwas immobilizedto biosensors coated with streptavidin Binding profiles weredisplayed by sensograms Global kinetic analyses were deter-mined using a 2 1 heterogeneous ligand model fit using R-linked analysis
233 HER2 Affinity Assay HER2 expressing cells SK-BR-3(ATCC HTB-30) were incubated in the presence of differentconcentrations of trastuzumab in McCoy-5A medium with10 FBS for 2 h at 37∘C HRP-conjugated goat anti-humanIgG was added to detect the trastuzumabndashSK-BR-3 complexafter 1 h of incubation at 37∘C using TMB as substrate for30min at room temperature Absorption was measured at450 nm Test results were expressed as the relative percentageof the EC
50from the dose-response curve of the biosimilar
trastuzumab with respect to the reference product
234 Antiproliferation Assay BT-474 cells (ATCC HTB-20)were seeded in DMEMmedia with 10 FBS 1 nonessentialamino acids and incubated at 37∘C Different concentrationsof trastuzumab were added with further incubation for 8days Crystal violet was added to stain the cells for 15minat room temperature followed by fixation with formaldehydeand water rising Acetic acid aqueous solution (33 vv) wasadded to remove the dye excess absorbance was measured at540 nm Test results were expressed as the relative percentage
BioMed Research International 3
Physicochemical characterization
Physical characterization
Functional assays
Comparability
Charge- CEX CZE HIC cIEF
Glycosylation- HILIC CZE
Degradation- CGE-NR
Purity- CGE-R SEC AUC CZE
Isoelectric point- cIEF
Identity- MS peptide mapping
SDS-PAGE WB
Size- SEC
Massradii- SEC-MALS MS
- FL TCSPCDSC CD HDX Ellman
Aggregation- ESZ SEC AUC
Biological activity- ADCC CDC
antiproliferation assayAffinity
- ITC BLI SPRELISA
Structure
Figure 1 Characterization strategy performed for Trastuzumab-Probiomed
Figure 2 Mirror plot of peptide mapping chromatograms obtained from RP-UPLC-UV for Trastuzumab-Probiomed (upper) and thereference product (lower)
of the EC50
from the dose-response curve of the biosimilartrastuzumab with respect to the reference product
3 Results and Discussion
Our characterization strategy (Figure 1) comprised a set ofstate-of-the-art analytical techniques planned for a hierarchi-cal study of a biosimilar trastuzumab using an orthogonalapproach CQAs were identified using a risk analysis consid-ering each of the physicochemical and functional propertiesthat may have an impact on efficacy (pharmacokineticsand pharmacodynamics) and safety (immunogenicity) oftrastuzumab (Table 1) [9ndash17] In this work only certainmethodologies were selected to depict a global overview
of the characterization study Hereafter CQAs were classi-fied by their physicochemical physical or biological natureand analyzed comparatively for a biosimilar trastuzumab(Trastuzumab-Probiomed) and its reference product
31 Physicochemical Properties The identity of Trastuzumab-Probiomed towards the reference product was determinedby the correspondence of their tryptic peptide mappings(Figure 2) MSMS analysis verified the amino acid sequenceof both products against the theoretical stated on the inven-tion patent of trastuzumab [28] unveiling a sequence match-ing of 998 and 993 for the heavy chain and 995 and995 for the light chain for both Trastuzumab-Probiomedand the reference product respectively (Figures 3 and 4)
Figure 3 Sequence coverage of the heavy and light chains of Trastuzumab-Probiomed obtained from the MSMS analysis
This correspondence was further confirmed by the analysesof the exact masses against the theoretical mass [28 29]for both whole and deglycosylated molecules (Tables 2 and3) The sequences coverage confirms that the amino acidsequence of Trastuzumab-Probiomed is identical to thereference product while the lt25Da observed differences inintact masses for the whole molecule below the expectedwidth of the isotopic pattern distribution of a mAb showin advance a comparable degree of heterogeneity due toposttranslational modifications in both products ultimatelyproducing an equivalent immunogenic response
Regarding glycan microheterogeneity which is known tocontribute to the correct folding and stability of a mAb itwas analyzed by CZE and HILI-UPLC Particularly highlymannosylated and sialylated glycoforms are reported toalter a mAb half-life in blood and are linked to potentialimmunogenic responses moreover effector functions can bealtered due to the presence of highly mannosylated bisectedand fucosylated glycoforms as a consequence of charge orsteric hindrances [10ndash12]
CZE analyses revealed that the glycan patterns ofTrastuzumab-Probiomed and the reference product arecomprised of the same principal glycoforms (Figure 5(a))showing amean relative abundance of galactosylated variantsof 6601 and 4957 plusmn 618 (CI 95) for Trastuzumab-Probiomed and the reference product respectively which isnot expected to have an impact on the functional propertiessince galactosylation has not been reported to alter themechanisms of action of mAbs as confirmed by the affinitiesand biological potency analyses discussed below Further
analysis by HILI-UPLC of the glycoforms identified ascritical for PK PD or immunogenicity (Table 1) revealedcomparable relative abundances of highly mannosylatedvariants being 200 plusmn 010 (CI 95) and 396 plusmn 045 (CI 95)for Trastuzumab-Probiomed and the reference productrespectively whereas the mean abundance for hybrid andsialylated variants was 475 plusmn 019 (CI 95) and 027 plusmn008 (CI 95) for the reference product and 295 plusmn 015 (CI95) and 106 plusmn 014 (CI 95) for Trastuzumab-Probiomedrespectively These results confirm similarity of the criticalglycoforms between Trastuzumab-Probiomed and thereference product thus similar PK and PD profiles and nodifferential immunogenicity response are expected
On the other hand charge heterogeneity evaluatedthrough cIEF analysis revealed that isoelectric points (pI) forthemain isoformwere 869plusmn 000 (CI 95) for Trastuzumab-Probiomed and 870 plusmn 001 (CI 95) for the referenceproduct in accordancewith the expected pI variations duringmanufacturing no larger than 02 units [15 16]The observedisoform-abundance-weighted pI values confirmed similarityof charge heterogeneity among products being 860 plusmn 001(CI 95) for Trastuzumab-Probiomed and 861 plusmn 001 (CI95) for the reference product It has been reported that onlychanges in one pI unit can significantly alter the therapeuticactivity of a mAb thus the observed variation is not expectedto affect the clinical behavior of Trastuzumab-Probiomedwith respect to the reference product
An orthogonal analytical technique for the evaluationof charge heterogeneity was CEX-UPLC which revealedthat the averaged abundances of the main acidic and basic
6 BioMed Research International
S C A A S G F N I K
S A D T S K N T A Y
A S T K G P S V F P
H T F P A V L Q S S
K S C D K T H T C P
H E D P E V K F N W
E Y K C K V S N K A
L V K G F Y P S D I
Q Q G N V F S C S V
I T C R A S Q D V N
F T L T I S S L Q P
S D E Q L K S G T A
S T Y S L S S T L T
D T Y I H W V R Q A
L Q M N S L R A E D
L A P S S K S T S G
P C P A P E L L G G
G L Y S L S S V V T
Y V D G V E V H N A
L P A P I E K T I S
A V E W E S N G Q P
M H E A L H N H Y T
T A V A W Y Q Q K P
E D F A T Y Y C Q Q
S V V C L L N N F Y
L S K A D Y E K H K
A RK
P G K G L E W V A R
T A V Y Y C S R W G
G T A A L G C L V K
V P S S S L G T Q T
P S V F L F P P K P
K T K P R E E Q Y N
K G Q P E P Q
E N N Y K T T P P V
Q K S L S L S P G K
G K A P K L L I Y S
H Y T T P P T F G Q
P R E A K V Q W K V
VY A C E V T H Q G
E V Q L V E S G G G
I Y P T N G Y T R Y
G D G F Y A M D Y W
D Y F P E P V T V S
Y I C N V N H K P S
K D T L M I S R T P
S T Y R V V S V L T
V Y T L P P S R E E
L D S D G S F F L Y
R
D I Q M T Q S P S S
A S F L Y S G V P S
G T K V E I K T V
D N A L Q S G N S Q
L S S P V T K S F N
L V Q P G G S L R L
A D S V K G R F T I
G Q G T L V T V S S
W N S G A L T S G V
N T K V D K K V E P
E V T C V V V D V S
V L H Q D W L N G K
M T K N Q V S L T C
S K L T V D K S R W
L S A S V G D R V T
R F S G S R S G T D
A A P S V F I F P P
E S V T E Q D S K D
RG E C
Control coverage () 995 Control unique coverage () 995
Figure 4 Sequence coverage of the heavy and light chains of the reference product obtained from the MSMS analysis
EU
G0F G1F
G1F998400
G2F
(min)
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
(a)
900
800
700
600
500
400
300
200
100
060 65 70 75 80 85 90
Temperature (∘C)
Fit (120583
J)
(b)
Figure 5 (a) Glycan profile for the reference product (upper) and Trastuzumab-Probiomed (lower) (b) Thermostability by DSC for thereference product (lower) and Trastuzumab-Probiomed (upper)
isoforms were within the same order of magnitude for bothproducts being the mean values of 570 332 and 98(119899 = 3) for Trastuzumab-Probiomed and 625 273and 103 (119899 = 3) for the reference product respectivelyFurthermore the results obtained after digestion with car-boxypeptidase B showed also a comparable content of basic
acidic and main isoforms among the two products with amain relative content of 164 306 and 530 (119899 = 3) forthe reference product and 88 378 and 534 (119899 = 3) forTrastuzumab-Probiomed respectively
After papain digestion the mean abundance of basicisoforms in the reference product (119899 = 3) was 38 for
the Fc fragment and 49 for the Fab fragment whereasfor Trastuzumab-Probiomed (119899 = 3) it was 42 for theFc fragment and 65 for the Fab fragment Regardingacidic isoforms the mean abundance was 33 for the Fcfragment and 167 for the Fab fragment of the referenceproduct while for Trastuzumab-Probiomed it was 37 forthe Fc fragment and 161 for the Fab fragment Finally theabundance of the Fc and Fab fragments was 267 and 446respectively for the reference product and for Trastuzumab-Probiomed the abundance of the Fc and Fab fragments was257 and 438 respectively
Overall the results from cIEF and CEX-UPLC show thatboth products exhibit comparable charge heterogeneitieseither as a whole molecule or as the fragments responsible forthe recognition and effector functions of trastuzumab thusno differences in functional activity should be expected
CGE-NR and SE-UPLC results demonstrated that bothproducts have a similar degree of purity (Tables 4 and 5)based on the relative content of monomer with respect tothe presence of aggregates and other degraded or truncatedisoforms It is known that protein aggregation can induceimmunogenicity although a small amount of aggregates isexpected this amount is likely to increase due to stressconditions that a mAb may undergo during its manufacturepurification formulation and shelf-life [9 30] Aggregationmay reveal new epitopes that potentially could stimulate theproduction of anti-drug antibodies (ADAs) resulting in theloss of activity immunogenic reactions or adverse effectsduring administration Likewise the presence of fragmentsor truncated forms coming from hydrolysis reactions couldnegatively impact on the safety and therapeutic effect of amAb [31 32] The content of aggregates and truncated forms
Table 4 Monomer content of trastuzumab by SE-UPLC and CGE-NR Variation is presented as confidence intervals at 95 (119899 = 3)
of Trastuzumab-Probiomed were lower than the limits estab-lished by the USP [29] and were comparable to the referenceproduct thus the risk of developing a different immunogenicresponse (differential immunogenicity) is diminished
32 Physical Properties Since the functionality of trastuz-umab is affected by its three-dimensional structure whichresults from its primary sequence and posttranslationalmod-ifications that alter its size mass folding and stability [8]we performed analyses to assess the spatial configuration ofTrastuzumab-Probiomed compared to its reference productTime correlated single photon counting analysis (TCSPC)was employed to evaluate the fluorescence lifetime (120591) whichdepends on the exposure of aromatic amino acids within theprotein thus demonstrating similarity when the results areobtained from comparative analyses [33ndash36] TCSPC resultsshowed that the averaged 120591 of Trastuzumab-Probiomed was343Eminus09 plusmn 139Eminus10 s (CI 95) while the averaged 120591 forthe reference product was 349Eminus09 plusmn 169Eminus11 s (CI 95)Regarding CD the obtained spectrograms were superimpos-able in both near- and far-UV regions (Figure 6) suggestingthat alpha helix beta sheets random coil disulfide bondsand aromatic amino acids are distributed in a comparablespatial arrangement Finally transition temperatures (119879
119898) of
Trastuzumab-Probiomed (119899 = 3) by DSC (Figure 5(b)) were704∘C 791∘C 810∘C and 825∘C whereas for the referenceproduct (119899 = 3) they were 705∘C 796∘C 812∘C and 827∘Cfor both products the CI at 95 was lt002∘C for all thetemperatures Collectively TCSPC CD andDSC determinedthat thermostability and secondary and tertiary structures ofTrastuzumab-Probiomed were comparable to the referenceproduct In particular thermostability results are indicative ofa proper protein folding of both products in their respective
8 BioMed Research International
Table 5 Relative abundance of trastuzumab subunits by CGE-R Variation is presented as confidence interval at 95 (119899 = 3)
HC heavy chain NGHC nonglycosylated heavy chain and LC light chain
7
0
minus10
minus17240 250 300 350
Wavelength (nm)
CD (m
deg
)
(a)
30
20
10
0
minus10
minus20200 220 240 260 280 300
Wavelength (nm)CD
(m d
eg)
(b)
Figure 6Analysis of the three-dimensional structure of trastuzumabbyCDofTrastuzumab-Probiomed (solid line) and the reference product(dotted line) in both near-UV region (a) and far-UV region (b)
3
2
1
1
0
10 100 1000 10000
OD
(450
nm)
Trastuzumab (ngmL)
(a)
10 100 1000 10000
Trastuzumab (ngmL)
285
235
185
135
085
035
OD
(540
nm)
(b)
Figure 7 Comparison of in vitro activity between Trastuzumab-Probiomed and the reference product (a) Curve of binding affinity to HER2(b) potency curve obtained from the antiproliferation assay the solid line corresponds to Trastuzumab-Probiomed while the dashed linecorresponds to the reference product
formulation This physicochemical and physical similarity isthe major contributor to equivalent biological and functionalresponses
33 Functional Properties The relative affinity of Trastuzum-ab-Probiomed towards its targetmoleculeHER2 (Figure 7(a)and Table 7) was evaluated with respect to the referenceproduct resulting in an averaged relative affinity of 977Thus it is expected that Trastuzumab-Probiomed can exert itsactivity through the reported mechanisms of action includ-ing HER2 downregulation prevention of the heterodimerformation initiation of Gl arrest induction of p27 andprevention of HER2 cleavage [37]
The main mechanisms of action rely on the affinityof the Fc fragment of trastuzumab towards Fc120574 receptorsFor instance Fc120574RIIIa present on effector cells such asmacrophages monocytes and natural killer cells activatesand induces ADCC mechanism against HER2-positive cells[37 38] Binding affinities towards Fc120574RIIIa were evalu-ated by ITC being the averaged affinity constants (119870
119886) of
261 plusmn 054E+06Mminus1 for Trastuzumab-Probiomed and 248 plusmn030E+06Mminus1 for the reference product (Table 6) Likewisethe mean dissociation constants (119870
119863) to FcRn which regu-
lates IgG catabolism were determined by BLI as 258Eminus07Mplusmn 102Eminus07M (CI 95) for Trastuzumab-Probiomed with arelative binding affinity of 1143 (119899 = 3) with respect to
BioMed Research International 9
Table 6 Affinity of trastuzumab to Fc120574RIIIa
Product Batch Affinity constant (119870119886
) toFc120574RIIIa (Mminus1)
Trastuzumab-Probiomed
TZPP14001 271119864 + 06
TZPP12002 286119864 + 06
TZPP12003 225119864 + 06
Reference productN35893 266119864 + 06
N35812 248119864 + 06
N36003 231119864 + 06
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
[1] M McCamish and G Woollett ldquoWorldwide experience withbiosimilar developmentrdquomAbs vol 3 no 2 pp 209ndash217 2011
[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
10 BioMed Research International
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
Figure 2 Mirror plot of peptide mapping chromatograms obtained from RP-UPLC-UV for Trastuzumab-Probiomed (upper) and thereference product (lower)
of the EC50
from the dose-response curve of the biosimilartrastuzumab with respect to the reference product
3 Results and Discussion
Our characterization strategy (Figure 1) comprised a set ofstate-of-the-art analytical techniques planned for a hierarchi-cal study of a biosimilar trastuzumab using an orthogonalapproach CQAs were identified using a risk analysis consid-ering each of the physicochemical and functional propertiesthat may have an impact on efficacy (pharmacokineticsand pharmacodynamics) and safety (immunogenicity) oftrastuzumab (Table 1) [9ndash17] In this work only certainmethodologies were selected to depict a global overview
of the characterization study Hereafter CQAs were classi-fied by their physicochemical physical or biological natureand analyzed comparatively for a biosimilar trastuzumab(Trastuzumab-Probiomed) and its reference product
31 Physicochemical Properties The identity of Trastuzumab-Probiomed towards the reference product was determinedby the correspondence of their tryptic peptide mappings(Figure 2) MSMS analysis verified the amino acid sequenceof both products against the theoretical stated on the inven-tion patent of trastuzumab [28] unveiling a sequence match-ing of 998 and 993 for the heavy chain and 995 and995 for the light chain for both Trastuzumab-Probiomedand the reference product respectively (Figures 3 and 4)
Figure 3 Sequence coverage of the heavy and light chains of Trastuzumab-Probiomed obtained from the MSMS analysis
This correspondence was further confirmed by the analysesof the exact masses against the theoretical mass [28 29]for both whole and deglycosylated molecules (Tables 2 and3) The sequences coverage confirms that the amino acidsequence of Trastuzumab-Probiomed is identical to thereference product while the lt25Da observed differences inintact masses for the whole molecule below the expectedwidth of the isotopic pattern distribution of a mAb showin advance a comparable degree of heterogeneity due toposttranslational modifications in both products ultimatelyproducing an equivalent immunogenic response
Regarding glycan microheterogeneity which is known tocontribute to the correct folding and stability of a mAb itwas analyzed by CZE and HILI-UPLC Particularly highlymannosylated and sialylated glycoforms are reported toalter a mAb half-life in blood and are linked to potentialimmunogenic responses moreover effector functions can bealtered due to the presence of highly mannosylated bisectedand fucosylated glycoforms as a consequence of charge orsteric hindrances [10ndash12]
CZE analyses revealed that the glycan patterns ofTrastuzumab-Probiomed and the reference product arecomprised of the same principal glycoforms (Figure 5(a))showing amean relative abundance of galactosylated variantsof 6601 and 4957 plusmn 618 (CI 95) for Trastuzumab-Probiomed and the reference product respectively which isnot expected to have an impact on the functional propertiessince galactosylation has not been reported to alter themechanisms of action of mAbs as confirmed by the affinitiesand biological potency analyses discussed below Further
analysis by HILI-UPLC of the glycoforms identified ascritical for PK PD or immunogenicity (Table 1) revealedcomparable relative abundances of highly mannosylatedvariants being 200 plusmn 010 (CI 95) and 396 plusmn 045 (CI 95)for Trastuzumab-Probiomed and the reference productrespectively whereas the mean abundance for hybrid andsialylated variants was 475 plusmn 019 (CI 95) and 027 plusmn008 (CI 95) for the reference product and 295 plusmn 015 (CI95) and 106 plusmn 014 (CI 95) for Trastuzumab-Probiomedrespectively These results confirm similarity of the criticalglycoforms between Trastuzumab-Probiomed and thereference product thus similar PK and PD profiles and nodifferential immunogenicity response are expected
On the other hand charge heterogeneity evaluatedthrough cIEF analysis revealed that isoelectric points (pI) forthemain isoformwere 869plusmn 000 (CI 95) for Trastuzumab-Probiomed and 870 plusmn 001 (CI 95) for the referenceproduct in accordancewith the expected pI variations duringmanufacturing no larger than 02 units [15 16]The observedisoform-abundance-weighted pI values confirmed similarityof charge heterogeneity among products being 860 plusmn 001(CI 95) for Trastuzumab-Probiomed and 861 plusmn 001 (CI95) for the reference product It has been reported that onlychanges in one pI unit can significantly alter the therapeuticactivity of a mAb thus the observed variation is not expectedto affect the clinical behavior of Trastuzumab-Probiomedwith respect to the reference product
An orthogonal analytical technique for the evaluationof charge heterogeneity was CEX-UPLC which revealedthat the averaged abundances of the main acidic and basic
6 BioMed Research International
S C A A S G F N I K
S A D T S K N T A Y
A S T K G P S V F P
H T F P A V L Q S S
K S C D K T H T C P
H E D P E V K F N W
E Y K C K V S N K A
L V K G F Y P S D I
Q Q G N V F S C S V
I T C R A S Q D V N
F T L T I S S L Q P
S D E Q L K S G T A
S T Y S L S S T L T
D T Y I H W V R Q A
L Q M N S L R A E D
L A P S S K S T S G
P C P A P E L L G G
G L Y S L S S V V T
Y V D G V E V H N A
L P A P I E K T I S
A V E W E S N G Q P
M H E A L H N H Y T
T A V A W Y Q Q K P
E D F A T Y Y C Q Q
S V V C L L N N F Y
L S K A D Y E K H K
A RK
P G K G L E W V A R
T A V Y Y C S R W G
G T A A L G C L V K
V P S S S L G T Q T
P S V F L F P P K P
K T K P R E E Q Y N
K G Q P E P Q
E N N Y K T T P P V
Q K S L S L S P G K
G K A P K L L I Y S
H Y T T P P T F G Q
P R E A K V Q W K V
VY A C E V T H Q G
E V Q L V E S G G G
I Y P T N G Y T R Y
G D G F Y A M D Y W
D Y F P E P V T V S
Y I C N V N H K P S
K D T L M I S R T P
S T Y R V V S V L T
V Y T L P P S R E E
L D S D G S F F L Y
R
D I Q M T Q S P S S
A S F L Y S G V P S
G T K V E I K T V
D N A L Q S G N S Q
L S S P V T K S F N
L V Q P G G S L R L
A D S V K G R F T I
G Q G T L V T V S S
W N S G A L T S G V
N T K V D K K V E P
E V T C V V V D V S
V L H Q D W L N G K
M T K N Q V S L T C
S K L T V D K S R W
L S A S V G D R V T
R F S G S R S G T D
A A P S V F I F P P
E S V T E Q D S K D
RG E C
Control coverage () 995 Control unique coverage () 995
Figure 4 Sequence coverage of the heavy and light chains of the reference product obtained from the MSMS analysis
EU
G0F G1F
G1F998400
G2F
(min)
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
(a)
900
800
700
600
500
400
300
200
100
060 65 70 75 80 85 90
Temperature (∘C)
Fit (120583
J)
(b)
Figure 5 (a) Glycan profile for the reference product (upper) and Trastuzumab-Probiomed (lower) (b) Thermostability by DSC for thereference product (lower) and Trastuzumab-Probiomed (upper)
isoforms were within the same order of magnitude for bothproducts being the mean values of 570 332 and 98(119899 = 3) for Trastuzumab-Probiomed and 625 273and 103 (119899 = 3) for the reference product respectivelyFurthermore the results obtained after digestion with car-boxypeptidase B showed also a comparable content of basic
acidic and main isoforms among the two products with amain relative content of 164 306 and 530 (119899 = 3) forthe reference product and 88 378 and 534 (119899 = 3) forTrastuzumab-Probiomed respectively
After papain digestion the mean abundance of basicisoforms in the reference product (119899 = 3) was 38 for
the Fc fragment and 49 for the Fab fragment whereasfor Trastuzumab-Probiomed (119899 = 3) it was 42 for theFc fragment and 65 for the Fab fragment Regardingacidic isoforms the mean abundance was 33 for the Fcfragment and 167 for the Fab fragment of the referenceproduct while for Trastuzumab-Probiomed it was 37 forthe Fc fragment and 161 for the Fab fragment Finally theabundance of the Fc and Fab fragments was 267 and 446respectively for the reference product and for Trastuzumab-Probiomed the abundance of the Fc and Fab fragments was257 and 438 respectively
Overall the results from cIEF and CEX-UPLC show thatboth products exhibit comparable charge heterogeneitieseither as a whole molecule or as the fragments responsible forthe recognition and effector functions of trastuzumab thusno differences in functional activity should be expected
CGE-NR and SE-UPLC results demonstrated that bothproducts have a similar degree of purity (Tables 4 and 5)based on the relative content of monomer with respect tothe presence of aggregates and other degraded or truncatedisoforms It is known that protein aggregation can induceimmunogenicity although a small amount of aggregates isexpected this amount is likely to increase due to stressconditions that a mAb may undergo during its manufacturepurification formulation and shelf-life [9 30] Aggregationmay reveal new epitopes that potentially could stimulate theproduction of anti-drug antibodies (ADAs) resulting in theloss of activity immunogenic reactions or adverse effectsduring administration Likewise the presence of fragmentsor truncated forms coming from hydrolysis reactions couldnegatively impact on the safety and therapeutic effect of amAb [31 32] The content of aggregates and truncated forms
Table 4 Monomer content of trastuzumab by SE-UPLC and CGE-NR Variation is presented as confidence intervals at 95 (119899 = 3)
of Trastuzumab-Probiomed were lower than the limits estab-lished by the USP [29] and were comparable to the referenceproduct thus the risk of developing a different immunogenicresponse (differential immunogenicity) is diminished
32 Physical Properties Since the functionality of trastuz-umab is affected by its three-dimensional structure whichresults from its primary sequence and posttranslationalmod-ifications that alter its size mass folding and stability [8]we performed analyses to assess the spatial configuration ofTrastuzumab-Probiomed compared to its reference productTime correlated single photon counting analysis (TCSPC)was employed to evaluate the fluorescence lifetime (120591) whichdepends on the exposure of aromatic amino acids within theprotein thus demonstrating similarity when the results areobtained from comparative analyses [33ndash36] TCSPC resultsshowed that the averaged 120591 of Trastuzumab-Probiomed was343Eminus09 plusmn 139Eminus10 s (CI 95) while the averaged 120591 forthe reference product was 349Eminus09 plusmn 169Eminus11 s (CI 95)Regarding CD the obtained spectrograms were superimpos-able in both near- and far-UV regions (Figure 6) suggestingthat alpha helix beta sheets random coil disulfide bondsand aromatic amino acids are distributed in a comparablespatial arrangement Finally transition temperatures (119879
119898) of
Trastuzumab-Probiomed (119899 = 3) by DSC (Figure 5(b)) were704∘C 791∘C 810∘C and 825∘C whereas for the referenceproduct (119899 = 3) they were 705∘C 796∘C 812∘C and 827∘Cfor both products the CI at 95 was lt002∘C for all thetemperatures Collectively TCSPC CD andDSC determinedthat thermostability and secondary and tertiary structures ofTrastuzumab-Probiomed were comparable to the referenceproduct In particular thermostability results are indicative ofa proper protein folding of both products in their respective
8 BioMed Research International
Table 5 Relative abundance of trastuzumab subunits by CGE-R Variation is presented as confidence interval at 95 (119899 = 3)
HC heavy chain NGHC nonglycosylated heavy chain and LC light chain
7
0
minus10
minus17240 250 300 350
Wavelength (nm)
CD (m
deg
)
(a)
30
20
10
0
minus10
minus20200 220 240 260 280 300
Wavelength (nm)CD
(m d
eg)
(b)
Figure 6Analysis of the three-dimensional structure of trastuzumabbyCDofTrastuzumab-Probiomed (solid line) and the reference product(dotted line) in both near-UV region (a) and far-UV region (b)
3
2
1
1
0
10 100 1000 10000
OD
(450
nm)
Trastuzumab (ngmL)
(a)
10 100 1000 10000
Trastuzumab (ngmL)
285
235
185
135
085
035
OD
(540
nm)
(b)
Figure 7 Comparison of in vitro activity between Trastuzumab-Probiomed and the reference product (a) Curve of binding affinity to HER2(b) potency curve obtained from the antiproliferation assay the solid line corresponds to Trastuzumab-Probiomed while the dashed linecorresponds to the reference product
formulation This physicochemical and physical similarity isthe major contributor to equivalent biological and functionalresponses
33 Functional Properties The relative affinity of Trastuzum-ab-Probiomed towards its targetmoleculeHER2 (Figure 7(a)and Table 7) was evaluated with respect to the referenceproduct resulting in an averaged relative affinity of 977Thus it is expected that Trastuzumab-Probiomed can exert itsactivity through the reported mechanisms of action includ-ing HER2 downregulation prevention of the heterodimerformation initiation of Gl arrest induction of p27 andprevention of HER2 cleavage [37]
The main mechanisms of action rely on the affinityof the Fc fragment of trastuzumab towards Fc120574 receptorsFor instance Fc120574RIIIa present on effector cells such asmacrophages monocytes and natural killer cells activatesand induces ADCC mechanism against HER2-positive cells[37 38] Binding affinities towards Fc120574RIIIa were evalu-ated by ITC being the averaged affinity constants (119870
119886) of
261 plusmn 054E+06Mminus1 for Trastuzumab-Probiomed and 248 plusmn030E+06Mminus1 for the reference product (Table 6) Likewisethe mean dissociation constants (119870
119863) to FcRn which regu-
lates IgG catabolism were determined by BLI as 258Eminus07Mplusmn 102Eminus07M (CI 95) for Trastuzumab-Probiomed with arelative binding affinity of 1143 (119899 = 3) with respect to
BioMed Research International 9
Table 6 Affinity of trastuzumab to Fc120574RIIIa
Product Batch Affinity constant (119870119886
) toFc120574RIIIa (Mminus1)
Trastuzumab-Probiomed
TZPP14001 271119864 + 06
TZPP12002 286119864 + 06
TZPP12003 225119864 + 06
Reference productN35893 266119864 + 06
N35812 248119864 + 06
N36003 231119864 + 06
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
[1] M McCamish and G Woollett ldquoWorldwide experience withbiosimilar developmentrdquomAbs vol 3 no 2 pp 209ndash217 2011
[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
10 BioMed Research International
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
Figure 3 Sequence coverage of the heavy and light chains of Trastuzumab-Probiomed obtained from the MSMS analysis
This correspondence was further confirmed by the analysesof the exact masses against the theoretical mass [28 29]for both whole and deglycosylated molecules (Tables 2 and3) The sequences coverage confirms that the amino acidsequence of Trastuzumab-Probiomed is identical to thereference product while the lt25Da observed differences inintact masses for the whole molecule below the expectedwidth of the isotopic pattern distribution of a mAb showin advance a comparable degree of heterogeneity due toposttranslational modifications in both products ultimatelyproducing an equivalent immunogenic response
Regarding glycan microheterogeneity which is known tocontribute to the correct folding and stability of a mAb itwas analyzed by CZE and HILI-UPLC Particularly highlymannosylated and sialylated glycoforms are reported toalter a mAb half-life in blood and are linked to potentialimmunogenic responses moreover effector functions can bealtered due to the presence of highly mannosylated bisectedand fucosylated glycoforms as a consequence of charge orsteric hindrances [10ndash12]
CZE analyses revealed that the glycan patterns ofTrastuzumab-Probiomed and the reference product arecomprised of the same principal glycoforms (Figure 5(a))showing amean relative abundance of galactosylated variantsof 6601 and 4957 plusmn 618 (CI 95) for Trastuzumab-Probiomed and the reference product respectively which isnot expected to have an impact on the functional propertiessince galactosylation has not been reported to alter themechanisms of action of mAbs as confirmed by the affinitiesand biological potency analyses discussed below Further
analysis by HILI-UPLC of the glycoforms identified ascritical for PK PD or immunogenicity (Table 1) revealedcomparable relative abundances of highly mannosylatedvariants being 200 plusmn 010 (CI 95) and 396 plusmn 045 (CI 95)for Trastuzumab-Probiomed and the reference productrespectively whereas the mean abundance for hybrid andsialylated variants was 475 plusmn 019 (CI 95) and 027 plusmn008 (CI 95) for the reference product and 295 plusmn 015 (CI95) and 106 plusmn 014 (CI 95) for Trastuzumab-Probiomedrespectively These results confirm similarity of the criticalglycoforms between Trastuzumab-Probiomed and thereference product thus similar PK and PD profiles and nodifferential immunogenicity response are expected
On the other hand charge heterogeneity evaluatedthrough cIEF analysis revealed that isoelectric points (pI) forthemain isoformwere 869plusmn 000 (CI 95) for Trastuzumab-Probiomed and 870 plusmn 001 (CI 95) for the referenceproduct in accordancewith the expected pI variations duringmanufacturing no larger than 02 units [15 16]The observedisoform-abundance-weighted pI values confirmed similarityof charge heterogeneity among products being 860 plusmn 001(CI 95) for Trastuzumab-Probiomed and 861 plusmn 001 (CI95) for the reference product It has been reported that onlychanges in one pI unit can significantly alter the therapeuticactivity of a mAb thus the observed variation is not expectedto affect the clinical behavior of Trastuzumab-Probiomedwith respect to the reference product
An orthogonal analytical technique for the evaluationof charge heterogeneity was CEX-UPLC which revealedthat the averaged abundances of the main acidic and basic
6 BioMed Research International
S C A A S G F N I K
S A D T S K N T A Y
A S T K G P S V F P
H T F P A V L Q S S
K S C D K T H T C P
H E D P E V K F N W
E Y K C K V S N K A
L V K G F Y P S D I
Q Q G N V F S C S V
I T C R A S Q D V N
F T L T I S S L Q P
S D E Q L K S G T A
S T Y S L S S T L T
D T Y I H W V R Q A
L Q M N S L R A E D
L A P S S K S T S G
P C P A P E L L G G
G L Y S L S S V V T
Y V D G V E V H N A
L P A P I E K T I S
A V E W E S N G Q P
M H E A L H N H Y T
T A V A W Y Q Q K P
E D F A T Y Y C Q Q
S V V C L L N N F Y
L S K A D Y E K H K
A RK
P G K G L E W V A R
T A V Y Y C S R W G
G T A A L G C L V K
V P S S S L G T Q T
P S V F L F P P K P
K T K P R E E Q Y N
K G Q P E P Q
E N N Y K T T P P V
Q K S L S L S P G K
G K A P K L L I Y S
H Y T T P P T F G Q
P R E A K V Q W K V
VY A C E V T H Q G
E V Q L V E S G G G
I Y P T N G Y T R Y
G D G F Y A M D Y W
D Y F P E P V T V S
Y I C N V N H K P S
K D T L M I S R T P
S T Y R V V S V L T
V Y T L P P S R E E
L D S D G S F F L Y
R
D I Q M T Q S P S S
A S F L Y S G V P S
G T K V E I K T V
D N A L Q S G N S Q
L S S P V T K S F N
L V Q P G G S L R L
A D S V K G R F T I
G Q G T L V T V S S
W N S G A L T S G V
N T K V D K K V E P
E V T C V V V D V S
V L H Q D W L N G K
M T K N Q V S L T C
S K L T V D K S R W
L S A S V G D R V T
R F S G S R S G T D
A A P S V F I F P P
E S V T E Q D S K D
RG E C
Control coverage () 995 Control unique coverage () 995
Figure 4 Sequence coverage of the heavy and light chains of the reference product obtained from the MSMS analysis
EU
G0F G1F
G1F998400
G2F
(min)
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
(a)
900
800
700
600
500
400
300
200
100
060 65 70 75 80 85 90
Temperature (∘C)
Fit (120583
J)
(b)
Figure 5 (a) Glycan profile for the reference product (upper) and Trastuzumab-Probiomed (lower) (b) Thermostability by DSC for thereference product (lower) and Trastuzumab-Probiomed (upper)
isoforms were within the same order of magnitude for bothproducts being the mean values of 570 332 and 98(119899 = 3) for Trastuzumab-Probiomed and 625 273and 103 (119899 = 3) for the reference product respectivelyFurthermore the results obtained after digestion with car-boxypeptidase B showed also a comparable content of basic
acidic and main isoforms among the two products with amain relative content of 164 306 and 530 (119899 = 3) forthe reference product and 88 378 and 534 (119899 = 3) forTrastuzumab-Probiomed respectively
After papain digestion the mean abundance of basicisoforms in the reference product (119899 = 3) was 38 for
the Fc fragment and 49 for the Fab fragment whereasfor Trastuzumab-Probiomed (119899 = 3) it was 42 for theFc fragment and 65 for the Fab fragment Regardingacidic isoforms the mean abundance was 33 for the Fcfragment and 167 for the Fab fragment of the referenceproduct while for Trastuzumab-Probiomed it was 37 forthe Fc fragment and 161 for the Fab fragment Finally theabundance of the Fc and Fab fragments was 267 and 446respectively for the reference product and for Trastuzumab-Probiomed the abundance of the Fc and Fab fragments was257 and 438 respectively
Overall the results from cIEF and CEX-UPLC show thatboth products exhibit comparable charge heterogeneitieseither as a whole molecule or as the fragments responsible forthe recognition and effector functions of trastuzumab thusno differences in functional activity should be expected
CGE-NR and SE-UPLC results demonstrated that bothproducts have a similar degree of purity (Tables 4 and 5)based on the relative content of monomer with respect tothe presence of aggregates and other degraded or truncatedisoforms It is known that protein aggregation can induceimmunogenicity although a small amount of aggregates isexpected this amount is likely to increase due to stressconditions that a mAb may undergo during its manufacturepurification formulation and shelf-life [9 30] Aggregationmay reveal new epitopes that potentially could stimulate theproduction of anti-drug antibodies (ADAs) resulting in theloss of activity immunogenic reactions or adverse effectsduring administration Likewise the presence of fragmentsor truncated forms coming from hydrolysis reactions couldnegatively impact on the safety and therapeutic effect of amAb [31 32] The content of aggregates and truncated forms
Table 4 Monomer content of trastuzumab by SE-UPLC and CGE-NR Variation is presented as confidence intervals at 95 (119899 = 3)
of Trastuzumab-Probiomed were lower than the limits estab-lished by the USP [29] and were comparable to the referenceproduct thus the risk of developing a different immunogenicresponse (differential immunogenicity) is diminished
32 Physical Properties Since the functionality of trastuz-umab is affected by its three-dimensional structure whichresults from its primary sequence and posttranslationalmod-ifications that alter its size mass folding and stability [8]we performed analyses to assess the spatial configuration ofTrastuzumab-Probiomed compared to its reference productTime correlated single photon counting analysis (TCSPC)was employed to evaluate the fluorescence lifetime (120591) whichdepends on the exposure of aromatic amino acids within theprotein thus demonstrating similarity when the results areobtained from comparative analyses [33ndash36] TCSPC resultsshowed that the averaged 120591 of Trastuzumab-Probiomed was343Eminus09 plusmn 139Eminus10 s (CI 95) while the averaged 120591 forthe reference product was 349Eminus09 plusmn 169Eminus11 s (CI 95)Regarding CD the obtained spectrograms were superimpos-able in both near- and far-UV regions (Figure 6) suggestingthat alpha helix beta sheets random coil disulfide bondsand aromatic amino acids are distributed in a comparablespatial arrangement Finally transition temperatures (119879
119898) of
Trastuzumab-Probiomed (119899 = 3) by DSC (Figure 5(b)) were704∘C 791∘C 810∘C and 825∘C whereas for the referenceproduct (119899 = 3) they were 705∘C 796∘C 812∘C and 827∘Cfor both products the CI at 95 was lt002∘C for all thetemperatures Collectively TCSPC CD andDSC determinedthat thermostability and secondary and tertiary structures ofTrastuzumab-Probiomed were comparable to the referenceproduct In particular thermostability results are indicative ofa proper protein folding of both products in their respective
8 BioMed Research International
Table 5 Relative abundance of trastuzumab subunits by CGE-R Variation is presented as confidence interval at 95 (119899 = 3)
HC heavy chain NGHC nonglycosylated heavy chain and LC light chain
7
0
minus10
minus17240 250 300 350
Wavelength (nm)
CD (m
deg
)
(a)
30
20
10
0
minus10
minus20200 220 240 260 280 300
Wavelength (nm)CD
(m d
eg)
(b)
Figure 6Analysis of the three-dimensional structure of trastuzumabbyCDofTrastuzumab-Probiomed (solid line) and the reference product(dotted line) in both near-UV region (a) and far-UV region (b)
3
2
1
1
0
10 100 1000 10000
OD
(450
nm)
Trastuzumab (ngmL)
(a)
10 100 1000 10000
Trastuzumab (ngmL)
285
235
185
135
085
035
OD
(540
nm)
(b)
Figure 7 Comparison of in vitro activity between Trastuzumab-Probiomed and the reference product (a) Curve of binding affinity to HER2(b) potency curve obtained from the antiproliferation assay the solid line corresponds to Trastuzumab-Probiomed while the dashed linecorresponds to the reference product
formulation This physicochemical and physical similarity isthe major contributor to equivalent biological and functionalresponses
33 Functional Properties The relative affinity of Trastuzum-ab-Probiomed towards its targetmoleculeHER2 (Figure 7(a)and Table 7) was evaluated with respect to the referenceproduct resulting in an averaged relative affinity of 977Thus it is expected that Trastuzumab-Probiomed can exert itsactivity through the reported mechanisms of action includ-ing HER2 downregulation prevention of the heterodimerformation initiation of Gl arrest induction of p27 andprevention of HER2 cleavage [37]
The main mechanisms of action rely on the affinityof the Fc fragment of trastuzumab towards Fc120574 receptorsFor instance Fc120574RIIIa present on effector cells such asmacrophages monocytes and natural killer cells activatesand induces ADCC mechanism against HER2-positive cells[37 38] Binding affinities towards Fc120574RIIIa were evalu-ated by ITC being the averaged affinity constants (119870
119886) of
261 plusmn 054E+06Mminus1 for Trastuzumab-Probiomed and 248 plusmn030E+06Mminus1 for the reference product (Table 6) Likewisethe mean dissociation constants (119870
119863) to FcRn which regu-
lates IgG catabolism were determined by BLI as 258Eminus07Mplusmn 102Eminus07M (CI 95) for Trastuzumab-Probiomed with arelative binding affinity of 1143 (119899 = 3) with respect to
BioMed Research International 9
Table 6 Affinity of trastuzumab to Fc120574RIIIa
Product Batch Affinity constant (119870119886
) toFc120574RIIIa (Mminus1)
Trastuzumab-Probiomed
TZPP14001 271119864 + 06
TZPP12002 286119864 + 06
TZPP12003 225119864 + 06
Reference productN35893 266119864 + 06
N35812 248119864 + 06
N36003 231119864 + 06
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
[1] M McCamish and G Woollett ldquoWorldwide experience withbiosimilar developmentrdquomAbs vol 3 no 2 pp 209ndash217 2011
[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
10 BioMed Research International
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
Figure 3 Sequence coverage of the heavy and light chains of Trastuzumab-Probiomed obtained from the MSMS analysis
This correspondence was further confirmed by the analysesof the exact masses against the theoretical mass [28 29]for both whole and deglycosylated molecules (Tables 2 and3) The sequences coverage confirms that the amino acidsequence of Trastuzumab-Probiomed is identical to thereference product while the lt25Da observed differences inintact masses for the whole molecule below the expectedwidth of the isotopic pattern distribution of a mAb showin advance a comparable degree of heterogeneity due toposttranslational modifications in both products ultimatelyproducing an equivalent immunogenic response
Regarding glycan microheterogeneity which is known tocontribute to the correct folding and stability of a mAb itwas analyzed by CZE and HILI-UPLC Particularly highlymannosylated and sialylated glycoforms are reported toalter a mAb half-life in blood and are linked to potentialimmunogenic responses moreover effector functions can bealtered due to the presence of highly mannosylated bisectedand fucosylated glycoforms as a consequence of charge orsteric hindrances [10ndash12]
CZE analyses revealed that the glycan patterns ofTrastuzumab-Probiomed and the reference product arecomprised of the same principal glycoforms (Figure 5(a))showing amean relative abundance of galactosylated variantsof 6601 and 4957 plusmn 618 (CI 95) for Trastuzumab-Probiomed and the reference product respectively which isnot expected to have an impact on the functional propertiessince galactosylation has not been reported to alter themechanisms of action of mAbs as confirmed by the affinitiesand biological potency analyses discussed below Further
analysis by HILI-UPLC of the glycoforms identified ascritical for PK PD or immunogenicity (Table 1) revealedcomparable relative abundances of highly mannosylatedvariants being 200 plusmn 010 (CI 95) and 396 plusmn 045 (CI 95)for Trastuzumab-Probiomed and the reference productrespectively whereas the mean abundance for hybrid andsialylated variants was 475 plusmn 019 (CI 95) and 027 plusmn008 (CI 95) for the reference product and 295 plusmn 015 (CI95) and 106 plusmn 014 (CI 95) for Trastuzumab-Probiomedrespectively These results confirm similarity of the criticalglycoforms between Trastuzumab-Probiomed and thereference product thus similar PK and PD profiles and nodifferential immunogenicity response are expected
On the other hand charge heterogeneity evaluatedthrough cIEF analysis revealed that isoelectric points (pI) forthemain isoformwere 869plusmn 000 (CI 95) for Trastuzumab-Probiomed and 870 plusmn 001 (CI 95) for the referenceproduct in accordancewith the expected pI variations duringmanufacturing no larger than 02 units [15 16]The observedisoform-abundance-weighted pI values confirmed similarityof charge heterogeneity among products being 860 plusmn 001(CI 95) for Trastuzumab-Probiomed and 861 plusmn 001 (CI95) for the reference product It has been reported that onlychanges in one pI unit can significantly alter the therapeuticactivity of a mAb thus the observed variation is not expectedto affect the clinical behavior of Trastuzumab-Probiomedwith respect to the reference product
An orthogonal analytical technique for the evaluationof charge heterogeneity was CEX-UPLC which revealedthat the averaged abundances of the main acidic and basic
6 BioMed Research International
S C A A S G F N I K
S A D T S K N T A Y
A S T K G P S V F P
H T F P A V L Q S S
K S C D K T H T C P
H E D P E V K F N W
E Y K C K V S N K A
L V K G F Y P S D I
Q Q G N V F S C S V
I T C R A S Q D V N
F T L T I S S L Q P
S D E Q L K S G T A
S T Y S L S S T L T
D T Y I H W V R Q A
L Q M N S L R A E D
L A P S S K S T S G
P C P A P E L L G G
G L Y S L S S V V T
Y V D G V E V H N A
L P A P I E K T I S
A V E W E S N G Q P
M H E A L H N H Y T
T A V A W Y Q Q K P
E D F A T Y Y C Q Q
S V V C L L N N F Y
L S K A D Y E K H K
A RK
P G K G L E W V A R
T A V Y Y C S R W G
G T A A L G C L V K
V P S S S L G T Q T
P S V F L F P P K P
K T K P R E E Q Y N
K G Q P E P Q
E N N Y K T T P P V
Q K S L S L S P G K
G K A P K L L I Y S
H Y T T P P T F G Q
P R E A K V Q W K V
VY A C E V T H Q G
E V Q L V E S G G G
I Y P T N G Y T R Y
G D G F Y A M D Y W
D Y F P E P V T V S
Y I C N V N H K P S
K D T L M I S R T P
S T Y R V V S V L T
V Y T L P P S R E E
L D S D G S F F L Y
R
D I Q M T Q S P S S
A S F L Y S G V P S
G T K V E I K T V
D N A L Q S G N S Q
L S S P V T K S F N
L V Q P G G S L R L
A D S V K G R F T I
G Q G T L V T V S S
W N S G A L T S G V
N T K V D K K V E P
E V T C V V V D V S
V L H Q D W L N G K
M T K N Q V S L T C
S K L T V D K S R W
L S A S V G D R V T
R F S G S R S G T D
A A P S V F I F P P
E S V T E Q D S K D
RG E C
Control coverage () 995 Control unique coverage () 995
Figure 4 Sequence coverage of the heavy and light chains of the reference product obtained from the MSMS analysis
EU
G0F G1F
G1F998400
G2F
(min)
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
(a)
900
800
700
600
500
400
300
200
100
060 65 70 75 80 85 90
Temperature (∘C)
Fit (120583
J)
(b)
Figure 5 (a) Glycan profile for the reference product (upper) and Trastuzumab-Probiomed (lower) (b) Thermostability by DSC for thereference product (lower) and Trastuzumab-Probiomed (upper)
isoforms were within the same order of magnitude for bothproducts being the mean values of 570 332 and 98(119899 = 3) for Trastuzumab-Probiomed and 625 273and 103 (119899 = 3) for the reference product respectivelyFurthermore the results obtained after digestion with car-boxypeptidase B showed also a comparable content of basic
acidic and main isoforms among the two products with amain relative content of 164 306 and 530 (119899 = 3) forthe reference product and 88 378 and 534 (119899 = 3) forTrastuzumab-Probiomed respectively
After papain digestion the mean abundance of basicisoforms in the reference product (119899 = 3) was 38 for
the Fc fragment and 49 for the Fab fragment whereasfor Trastuzumab-Probiomed (119899 = 3) it was 42 for theFc fragment and 65 for the Fab fragment Regardingacidic isoforms the mean abundance was 33 for the Fcfragment and 167 for the Fab fragment of the referenceproduct while for Trastuzumab-Probiomed it was 37 forthe Fc fragment and 161 for the Fab fragment Finally theabundance of the Fc and Fab fragments was 267 and 446respectively for the reference product and for Trastuzumab-Probiomed the abundance of the Fc and Fab fragments was257 and 438 respectively
Overall the results from cIEF and CEX-UPLC show thatboth products exhibit comparable charge heterogeneitieseither as a whole molecule or as the fragments responsible forthe recognition and effector functions of trastuzumab thusno differences in functional activity should be expected
CGE-NR and SE-UPLC results demonstrated that bothproducts have a similar degree of purity (Tables 4 and 5)based on the relative content of monomer with respect tothe presence of aggregates and other degraded or truncatedisoforms It is known that protein aggregation can induceimmunogenicity although a small amount of aggregates isexpected this amount is likely to increase due to stressconditions that a mAb may undergo during its manufacturepurification formulation and shelf-life [9 30] Aggregationmay reveal new epitopes that potentially could stimulate theproduction of anti-drug antibodies (ADAs) resulting in theloss of activity immunogenic reactions or adverse effectsduring administration Likewise the presence of fragmentsor truncated forms coming from hydrolysis reactions couldnegatively impact on the safety and therapeutic effect of amAb [31 32] The content of aggregates and truncated forms
Table 4 Monomer content of trastuzumab by SE-UPLC and CGE-NR Variation is presented as confidence intervals at 95 (119899 = 3)
of Trastuzumab-Probiomed were lower than the limits estab-lished by the USP [29] and were comparable to the referenceproduct thus the risk of developing a different immunogenicresponse (differential immunogenicity) is diminished
32 Physical Properties Since the functionality of trastuz-umab is affected by its three-dimensional structure whichresults from its primary sequence and posttranslationalmod-ifications that alter its size mass folding and stability [8]we performed analyses to assess the spatial configuration ofTrastuzumab-Probiomed compared to its reference productTime correlated single photon counting analysis (TCSPC)was employed to evaluate the fluorescence lifetime (120591) whichdepends on the exposure of aromatic amino acids within theprotein thus demonstrating similarity when the results areobtained from comparative analyses [33ndash36] TCSPC resultsshowed that the averaged 120591 of Trastuzumab-Probiomed was343Eminus09 plusmn 139Eminus10 s (CI 95) while the averaged 120591 forthe reference product was 349Eminus09 plusmn 169Eminus11 s (CI 95)Regarding CD the obtained spectrograms were superimpos-able in both near- and far-UV regions (Figure 6) suggestingthat alpha helix beta sheets random coil disulfide bondsand aromatic amino acids are distributed in a comparablespatial arrangement Finally transition temperatures (119879
119898) of
Trastuzumab-Probiomed (119899 = 3) by DSC (Figure 5(b)) were704∘C 791∘C 810∘C and 825∘C whereas for the referenceproduct (119899 = 3) they were 705∘C 796∘C 812∘C and 827∘Cfor both products the CI at 95 was lt002∘C for all thetemperatures Collectively TCSPC CD andDSC determinedthat thermostability and secondary and tertiary structures ofTrastuzumab-Probiomed were comparable to the referenceproduct In particular thermostability results are indicative ofa proper protein folding of both products in their respective
8 BioMed Research International
Table 5 Relative abundance of trastuzumab subunits by CGE-R Variation is presented as confidence interval at 95 (119899 = 3)
HC heavy chain NGHC nonglycosylated heavy chain and LC light chain
7
0
minus10
minus17240 250 300 350
Wavelength (nm)
CD (m
deg
)
(a)
30
20
10
0
minus10
minus20200 220 240 260 280 300
Wavelength (nm)CD
(m d
eg)
(b)
Figure 6Analysis of the three-dimensional structure of trastuzumabbyCDofTrastuzumab-Probiomed (solid line) and the reference product(dotted line) in both near-UV region (a) and far-UV region (b)
3
2
1
1
0
10 100 1000 10000
OD
(450
nm)
Trastuzumab (ngmL)
(a)
10 100 1000 10000
Trastuzumab (ngmL)
285
235
185
135
085
035
OD
(540
nm)
(b)
Figure 7 Comparison of in vitro activity between Trastuzumab-Probiomed and the reference product (a) Curve of binding affinity to HER2(b) potency curve obtained from the antiproliferation assay the solid line corresponds to Trastuzumab-Probiomed while the dashed linecorresponds to the reference product
formulation This physicochemical and physical similarity isthe major contributor to equivalent biological and functionalresponses
33 Functional Properties The relative affinity of Trastuzum-ab-Probiomed towards its targetmoleculeHER2 (Figure 7(a)and Table 7) was evaluated with respect to the referenceproduct resulting in an averaged relative affinity of 977Thus it is expected that Trastuzumab-Probiomed can exert itsactivity through the reported mechanisms of action includ-ing HER2 downregulation prevention of the heterodimerformation initiation of Gl arrest induction of p27 andprevention of HER2 cleavage [37]
The main mechanisms of action rely on the affinityof the Fc fragment of trastuzumab towards Fc120574 receptorsFor instance Fc120574RIIIa present on effector cells such asmacrophages monocytes and natural killer cells activatesand induces ADCC mechanism against HER2-positive cells[37 38] Binding affinities towards Fc120574RIIIa were evalu-ated by ITC being the averaged affinity constants (119870
119886) of
261 plusmn 054E+06Mminus1 for Trastuzumab-Probiomed and 248 plusmn030E+06Mminus1 for the reference product (Table 6) Likewisethe mean dissociation constants (119870
119863) to FcRn which regu-
lates IgG catabolism were determined by BLI as 258Eminus07Mplusmn 102Eminus07M (CI 95) for Trastuzumab-Probiomed with arelative binding affinity of 1143 (119899 = 3) with respect to
BioMed Research International 9
Table 6 Affinity of trastuzumab to Fc120574RIIIa
Product Batch Affinity constant (119870119886
) toFc120574RIIIa (Mminus1)
Trastuzumab-Probiomed
TZPP14001 271119864 + 06
TZPP12002 286119864 + 06
TZPP12003 225119864 + 06
Reference productN35893 266119864 + 06
N35812 248119864 + 06
N36003 231119864 + 06
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
[1] M McCamish and G Woollett ldquoWorldwide experience withbiosimilar developmentrdquomAbs vol 3 no 2 pp 209ndash217 2011
[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
10 BioMed Research International
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
Figure 4 Sequence coverage of the heavy and light chains of the reference product obtained from the MSMS analysis
EU
G0F G1F
G1F998400
G2F
(min)
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
(a)
900
800
700
600
500
400
300
200
100
060 65 70 75 80 85 90
Temperature (∘C)
Fit (120583
J)
(b)
Figure 5 (a) Glycan profile for the reference product (upper) and Trastuzumab-Probiomed (lower) (b) Thermostability by DSC for thereference product (lower) and Trastuzumab-Probiomed (upper)
isoforms were within the same order of magnitude for bothproducts being the mean values of 570 332 and 98(119899 = 3) for Trastuzumab-Probiomed and 625 273and 103 (119899 = 3) for the reference product respectivelyFurthermore the results obtained after digestion with car-boxypeptidase B showed also a comparable content of basic
acidic and main isoforms among the two products with amain relative content of 164 306 and 530 (119899 = 3) forthe reference product and 88 378 and 534 (119899 = 3) forTrastuzumab-Probiomed respectively
After papain digestion the mean abundance of basicisoforms in the reference product (119899 = 3) was 38 for
the Fc fragment and 49 for the Fab fragment whereasfor Trastuzumab-Probiomed (119899 = 3) it was 42 for theFc fragment and 65 for the Fab fragment Regardingacidic isoforms the mean abundance was 33 for the Fcfragment and 167 for the Fab fragment of the referenceproduct while for Trastuzumab-Probiomed it was 37 forthe Fc fragment and 161 for the Fab fragment Finally theabundance of the Fc and Fab fragments was 267 and 446respectively for the reference product and for Trastuzumab-Probiomed the abundance of the Fc and Fab fragments was257 and 438 respectively
Overall the results from cIEF and CEX-UPLC show thatboth products exhibit comparable charge heterogeneitieseither as a whole molecule or as the fragments responsible forthe recognition and effector functions of trastuzumab thusno differences in functional activity should be expected
CGE-NR and SE-UPLC results demonstrated that bothproducts have a similar degree of purity (Tables 4 and 5)based on the relative content of monomer with respect tothe presence of aggregates and other degraded or truncatedisoforms It is known that protein aggregation can induceimmunogenicity although a small amount of aggregates isexpected this amount is likely to increase due to stressconditions that a mAb may undergo during its manufacturepurification formulation and shelf-life [9 30] Aggregationmay reveal new epitopes that potentially could stimulate theproduction of anti-drug antibodies (ADAs) resulting in theloss of activity immunogenic reactions or adverse effectsduring administration Likewise the presence of fragmentsor truncated forms coming from hydrolysis reactions couldnegatively impact on the safety and therapeutic effect of amAb [31 32] The content of aggregates and truncated forms
Table 4 Monomer content of trastuzumab by SE-UPLC and CGE-NR Variation is presented as confidence intervals at 95 (119899 = 3)
of Trastuzumab-Probiomed were lower than the limits estab-lished by the USP [29] and were comparable to the referenceproduct thus the risk of developing a different immunogenicresponse (differential immunogenicity) is diminished
32 Physical Properties Since the functionality of trastuz-umab is affected by its three-dimensional structure whichresults from its primary sequence and posttranslationalmod-ifications that alter its size mass folding and stability [8]we performed analyses to assess the spatial configuration ofTrastuzumab-Probiomed compared to its reference productTime correlated single photon counting analysis (TCSPC)was employed to evaluate the fluorescence lifetime (120591) whichdepends on the exposure of aromatic amino acids within theprotein thus demonstrating similarity when the results areobtained from comparative analyses [33ndash36] TCSPC resultsshowed that the averaged 120591 of Trastuzumab-Probiomed was343Eminus09 plusmn 139Eminus10 s (CI 95) while the averaged 120591 forthe reference product was 349Eminus09 plusmn 169Eminus11 s (CI 95)Regarding CD the obtained spectrograms were superimpos-able in both near- and far-UV regions (Figure 6) suggestingthat alpha helix beta sheets random coil disulfide bondsand aromatic amino acids are distributed in a comparablespatial arrangement Finally transition temperatures (119879
119898) of
Trastuzumab-Probiomed (119899 = 3) by DSC (Figure 5(b)) were704∘C 791∘C 810∘C and 825∘C whereas for the referenceproduct (119899 = 3) they were 705∘C 796∘C 812∘C and 827∘Cfor both products the CI at 95 was lt002∘C for all thetemperatures Collectively TCSPC CD andDSC determinedthat thermostability and secondary and tertiary structures ofTrastuzumab-Probiomed were comparable to the referenceproduct In particular thermostability results are indicative ofa proper protein folding of both products in their respective
8 BioMed Research International
Table 5 Relative abundance of trastuzumab subunits by CGE-R Variation is presented as confidence interval at 95 (119899 = 3)
HC heavy chain NGHC nonglycosylated heavy chain and LC light chain
7
0
minus10
minus17240 250 300 350
Wavelength (nm)
CD (m
deg
)
(a)
30
20
10
0
minus10
minus20200 220 240 260 280 300
Wavelength (nm)CD
(m d
eg)
(b)
Figure 6Analysis of the three-dimensional structure of trastuzumabbyCDofTrastuzumab-Probiomed (solid line) and the reference product(dotted line) in both near-UV region (a) and far-UV region (b)
3
2
1
1
0
10 100 1000 10000
OD
(450
nm)
Trastuzumab (ngmL)
(a)
10 100 1000 10000
Trastuzumab (ngmL)
285
235
185
135
085
035
OD
(540
nm)
(b)
Figure 7 Comparison of in vitro activity between Trastuzumab-Probiomed and the reference product (a) Curve of binding affinity to HER2(b) potency curve obtained from the antiproliferation assay the solid line corresponds to Trastuzumab-Probiomed while the dashed linecorresponds to the reference product
formulation This physicochemical and physical similarity isthe major contributor to equivalent biological and functionalresponses
33 Functional Properties The relative affinity of Trastuzum-ab-Probiomed towards its targetmoleculeHER2 (Figure 7(a)and Table 7) was evaluated with respect to the referenceproduct resulting in an averaged relative affinity of 977Thus it is expected that Trastuzumab-Probiomed can exert itsactivity through the reported mechanisms of action includ-ing HER2 downregulation prevention of the heterodimerformation initiation of Gl arrest induction of p27 andprevention of HER2 cleavage [37]
The main mechanisms of action rely on the affinityof the Fc fragment of trastuzumab towards Fc120574 receptorsFor instance Fc120574RIIIa present on effector cells such asmacrophages monocytes and natural killer cells activatesand induces ADCC mechanism against HER2-positive cells[37 38] Binding affinities towards Fc120574RIIIa were evalu-ated by ITC being the averaged affinity constants (119870
119886) of
261 plusmn 054E+06Mminus1 for Trastuzumab-Probiomed and 248 plusmn030E+06Mminus1 for the reference product (Table 6) Likewisethe mean dissociation constants (119870
119863) to FcRn which regu-
lates IgG catabolism were determined by BLI as 258Eminus07Mplusmn 102Eminus07M (CI 95) for Trastuzumab-Probiomed with arelative binding affinity of 1143 (119899 = 3) with respect to
BioMed Research International 9
Table 6 Affinity of trastuzumab to Fc120574RIIIa
Product Batch Affinity constant (119870119886
) toFc120574RIIIa (Mminus1)
Trastuzumab-Probiomed
TZPP14001 271119864 + 06
TZPP12002 286119864 + 06
TZPP12003 225119864 + 06
Reference productN35893 266119864 + 06
N35812 248119864 + 06
N36003 231119864 + 06
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
[1] M McCamish and G Woollett ldquoWorldwide experience withbiosimilar developmentrdquomAbs vol 3 no 2 pp 209ndash217 2011
[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
10 BioMed Research International
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
the Fc fragment and 49 for the Fab fragment whereasfor Trastuzumab-Probiomed (119899 = 3) it was 42 for theFc fragment and 65 for the Fab fragment Regardingacidic isoforms the mean abundance was 33 for the Fcfragment and 167 for the Fab fragment of the referenceproduct while for Trastuzumab-Probiomed it was 37 forthe Fc fragment and 161 for the Fab fragment Finally theabundance of the Fc and Fab fragments was 267 and 446respectively for the reference product and for Trastuzumab-Probiomed the abundance of the Fc and Fab fragments was257 and 438 respectively
Overall the results from cIEF and CEX-UPLC show thatboth products exhibit comparable charge heterogeneitieseither as a whole molecule or as the fragments responsible forthe recognition and effector functions of trastuzumab thusno differences in functional activity should be expected
CGE-NR and SE-UPLC results demonstrated that bothproducts have a similar degree of purity (Tables 4 and 5)based on the relative content of monomer with respect tothe presence of aggregates and other degraded or truncatedisoforms It is known that protein aggregation can induceimmunogenicity although a small amount of aggregates isexpected this amount is likely to increase due to stressconditions that a mAb may undergo during its manufacturepurification formulation and shelf-life [9 30] Aggregationmay reveal new epitopes that potentially could stimulate theproduction of anti-drug antibodies (ADAs) resulting in theloss of activity immunogenic reactions or adverse effectsduring administration Likewise the presence of fragmentsor truncated forms coming from hydrolysis reactions couldnegatively impact on the safety and therapeutic effect of amAb [31 32] The content of aggregates and truncated forms
Table 4 Monomer content of trastuzumab by SE-UPLC and CGE-NR Variation is presented as confidence intervals at 95 (119899 = 3)
of Trastuzumab-Probiomed were lower than the limits estab-lished by the USP [29] and were comparable to the referenceproduct thus the risk of developing a different immunogenicresponse (differential immunogenicity) is diminished
32 Physical Properties Since the functionality of trastuz-umab is affected by its three-dimensional structure whichresults from its primary sequence and posttranslationalmod-ifications that alter its size mass folding and stability [8]we performed analyses to assess the spatial configuration ofTrastuzumab-Probiomed compared to its reference productTime correlated single photon counting analysis (TCSPC)was employed to evaluate the fluorescence lifetime (120591) whichdepends on the exposure of aromatic amino acids within theprotein thus demonstrating similarity when the results areobtained from comparative analyses [33ndash36] TCSPC resultsshowed that the averaged 120591 of Trastuzumab-Probiomed was343Eminus09 plusmn 139Eminus10 s (CI 95) while the averaged 120591 forthe reference product was 349Eminus09 plusmn 169Eminus11 s (CI 95)Regarding CD the obtained spectrograms were superimpos-able in both near- and far-UV regions (Figure 6) suggestingthat alpha helix beta sheets random coil disulfide bondsand aromatic amino acids are distributed in a comparablespatial arrangement Finally transition temperatures (119879
119898) of
Trastuzumab-Probiomed (119899 = 3) by DSC (Figure 5(b)) were704∘C 791∘C 810∘C and 825∘C whereas for the referenceproduct (119899 = 3) they were 705∘C 796∘C 812∘C and 827∘Cfor both products the CI at 95 was lt002∘C for all thetemperatures Collectively TCSPC CD andDSC determinedthat thermostability and secondary and tertiary structures ofTrastuzumab-Probiomed were comparable to the referenceproduct In particular thermostability results are indicative ofa proper protein folding of both products in their respective
8 BioMed Research International
Table 5 Relative abundance of trastuzumab subunits by CGE-R Variation is presented as confidence interval at 95 (119899 = 3)
HC heavy chain NGHC nonglycosylated heavy chain and LC light chain
7
0
minus10
minus17240 250 300 350
Wavelength (nm)
CD (m
deg
)
(a)
30
20
10
0
minus10
minus20200 220 240 260 280 300
Wavelength (nm)CD
(m d
eg)
(b)
Figure 6Analysis of the three-dimensional structure of trastuzumabbyCDofTrastuzumab-Probiomed (solid line) and the reference product(dotted line) in both near-UV region (a) and far-UV region (b)
3
2
1
1
0
10 100 1000 10000
OD
(450
nm)
Trastuzumab (ngmL)
(a)
10 100 1000 10000
Trastuzumab (ngmL)
285
235
185
135
085
035
OD
(540
nm)
(b)
Figure 7 Comparison of in vitro activity between Trastuzumab-Probiomed and the reference product (a) Curve of binding affinity to HER2(b) potency curve obtained from the antiproliferation assay the solid line corresponds to Trastuzumab-Probiomed while the dashed linecorresponds to the reference product
formulation This physicochemical and physical similarity isthe major contributor to equivalent biological and functionalresponses
33 Functional Properties The relative affinity of Trastuzum-ab-Probiomed towards its targetmoleculeHER2 (Figure 7(a)and Table 7) was evaluated with respect to the referenceproduct resulting in an averaged relative affinity of 977Thus it is expected that Trastuzumab-Probiomed can exert itsactivity through the reported mechanisms of action includ-ing HER2 downregulation prevention of the heterodimerformation initiation of Gl arrest induction of p27 andprevention of HER2 cleavage [37]
The main mechanisms of action rely on the affinityof the Fc fragment of trastuzumab towards Fc120574 receptorsFor instance Fc120574RIIIa present on effector cells such asmacrophages monocytes and natural killer cells activatesand induces ADCC mechanism against HER2-positive cells[37 38] Binding affinities towards Fc120574RIIIa were evalu-ated by ITC being the averaged affinity constants (119870
119886) of
261 plusmn 054E+06Mminus1 for Trastuzumab-Probiomed and 248 plusmn030E+06Mminus1 for the reference product (Table 6) Likewisethe mean dissociation constants (119870
119863) to FcRn which regu-
lates IgG catabolism were determined by BLI as 258Eminus07Mplusmn 102Eminus07M (CI 95) for Trastuzumab-Probiomed with arelative binding affinity of 1143 (119899 = 3) with respect to
BioMed Research International 9
Table 6 Affinity of trastuzumab to Fc120574RIIIa
Product Batch Affinity constant (119870119886
) toFc120574RIIIa (Mminus1)
Trastuzumab-Probiomed
TZPP14001 271119864 + 06
TZPP12002 286119864 + 06
TZPP12003 225119864 + 06
Reference productN35893 266119864 + 06
N35812 248119864 + 06
N36003 231119864 + 06
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
[1] M McCamish and G Woollett ldquoWorldwide experience withbiosimilar developmentrdquomAbs vol 3 no 2 pp 209ndash217 2011
[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
10 BioMed Research International
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
HC heavy chain NGHC nonglycosylated heavy chain and LC light chain
7
0
minus10
minus17240 250 300 350
Wavelength (nm)
CD (m
deg
)
(a)
30
20
10
0
minus10
minus20200 220 240 260 280 300
Wavelength (nm)CD
(m d
eg)
(b)
Figure 6Analysis of the three-dimensional structure of trastuzumabbyCDofTrastuzumab-Probiomed (solid line) and the reference product(dotted line) in both near-UV region (a) and far-UV region (b)
3
2
1
1
0
10 100 1000 10000
OD
(450
nm)
Trastuzumab (ngmL)
(a)
10 100 1000 10000
Trastuzumab (ngmL)
285
235
185
135
085
035
OD
(540
nm)
(b)
Figure 7 Comparison of in vitro activity between Trastuzumab-Probiomed and the reference product (a) Curve of binding affinity to HER2(b) potency curve obtained from the antiproliferation assay the solid line corresponds to Trastuzumab-Probiomed while the dashed linecorresponds to the reference product
formulation This physicochemical and physical similarity isthe major contributor to equivalent biological and functionalresponses
33 Functional Properties The relative affinity of Trastuzum-ab-Probiomed towards its targetmoleculeHER2 (Figure 7(a)and Table 7) was evaluated with respect to the referenceproduct resulting in an averaged relative affinity of 977Thus it is expected that Trastuzumab-Probiomed can exert itsactivity through the reported mechanisms of action includ-ing HER2 downregulation prevention of the heterodimerformation initiation of Gl arrest induction of p27 andprevention of HER2 cleavage [37]
The main mechanisms of action rely on the affinityof the Fc fragment of trastuzumab towards Fc120574 receptorsFor instance Fc120574RIIIa present on effector cells such asmacrophages monocytes and natural killer cells activatesand induces ADCC mechanism against HER2-positive cells[37 38] Binding affinities towards Fc120574RIIIa were evalu-ated by ITC being the averaged affinity constants (119870
119886) of
261 plusmn 054E+06Mminus1 for Trastuzumab-Probiomed and 248 plusmn030E+06Mminus1 for the reference product (Table 6) Likewisethe mean dissociation constants (119870
119863) to FcRn which regu-
lates IgG catabolism were determined by BLI as 258Eminus07Mplusmn 102Eminus07M (CI 95) for Trastuzumab-Probiomed with arelative binding affinity of 1143 (119899 = 3) with respect to
BioMed Research International 9
Table 6 Affinity of trastuzumab to Fc120574RIIIa
Product Batch Affinity constant (119870119886
) toFc120574RIIIa (Mminus1)
Trastuzumab-Probiomed
TZPP14001 271119864 + 06
TZPP12002 286119864 + 06
TZPP12003 225119864 + 06
Reference productN35893 266119864 + 06
N35812 248119864 + 06
N36003 231119864 + 06
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
[1] M McCamish and G Woollett ldquoWorldwide experience withbiosimilar developmentrdquomAbs vol 3 no 2 pp 209ndash217 2011
[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
10 BioMed Research International
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
Table 7 Binding affinity of trastuzumab to the epidermal growthfactor receptor (HER2)
Product Batch Relative affinity()
Trastuzumab-Probiomed
TZPP11001 98TZPP12004 98TZPP12003 97
Reference productN3654 119N36263 111N36443 112
the reference product Based on these results no differencesin the half-life in blood are expected
The overall in vitro activity was tested betweenTrastuzumab-Probiomed and the reference product with anantiproliferation assay (Figure 7(b)) which demonstratedthat both products have the same potency to deplete HER2-positive cells being the mean relative potencies towards thereference product of 105 103 and 110 for three differentbatches of Trastuzumab-Probiomed demonstrating thatsimilarity on physicochemical and physical critical qualityattributes results in a comparable biological potency
4 Conclusions
During the development of a biosimilar an extended char-acterization of its physicochemical and functional propertiesis required to gain a strong knowledge of its CQAs Thisallows the establishment of in-process control strategies andquality specifications to ensure batch-to-batch consistency inorder to obtain the desired product despite the fact that ithas been produced using a different manufacturing processwith respect to the reference product In addition the use oforthogonal methods during a comparability study provides aglobal overview of the molecule and confirms the observedresults on relevant modifications Here it was demonstratedthat similarity between the critical physicochemical attributesresulted in comparable biological properties
The observed physicochemical and functional similaritybetween products as part of the totality-of-the-evidencescheme will determine the extent of upcoming nonclinicaland clinical studies considering that it diminishes the uncer-tainty of exhibiting different pharmacological profiles
Conflict of Interests
Carlos A Lopez-MoralesMariana PMiranda-Hernandez LCarmina Juarez-Bayardo Nancy D Ramırez-Ibanez AlexisJ Romero-Dıaz Nelly Pina-Lara Nestor O Perez Luis FFlores-Ortiz and Emilio Medina-Rivero are employees ofProbiomed SA de CV which is developing manufacturingandmarketing biosimilar products Vıctor RCampos-Garcıadeclared no conflict of interests
Acknowledgment
Financial support was provided by the National Councilfor Science and Technology (CONACYT) Mexico GrantFINNOVA 174102 without participation in the design of thestudy
References
[1] M McCamish and G Woollett ldquoWorldwide experience withbiosimilar developmentrdquomAbs vol 3 no 2 pp 209ndash217 2011
[2] ICH ldquoICH Q5E comparability of BiotechnologicalBiologicalproducts subject to changes in their manufacturing processrdquo inProceedings of the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals forHuman Use November 2004
[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct (Draft) FDA Rockville Md USA 2012
[4] Food and Drug Administration Guidance for Industry QualityConsiderations in Demonstrating Biosimilarity to a ReferenceProtein Product (Draft) FDA Rockville Md USA 2012
[5] Food and Drug Administration Guidance for Industry ClinicalPharmacology Data to Support a Demostration of Biosimilarityto a Reference Product draft FDA Rockville Md USA 2014
[6] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014
[7] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013
[8] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012
[9] G Shankar C Pendley and K E Stein ldquoA risk-based bioanalyt-ical strategy for the assessment of antibody immune responsesagainst biological drugsrdquo Nature Biotechnology vol 25 no 5pp 555ndash561 2007
[10] W S Putnam S Prabhu Y ZhengM Subramanyam andY-MC Wang ldquoPharmacokinetic pharmacodynamic and immuno-genicity comparability assessment strategies for monoclonalantibodiesrdquoTrends in Biotechnology vol 28 no 10 pp 509ndash5162010
[11] L Liu A Stadheim L Hamuro et al ldquoPharmacokinetics ofIgG1 monoclonal antibodies produced in humanized Pichiapastoris with specific glycoforms a comparative study withCHO produced materialsrdquo Biologicals vol 39 no 4 pp 205ndash210 2011
10 BioMed Research International
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
[12] T T Junttila K Parsons C Olsson et al ldquoSuperior in vivoefficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancerrdquo Cancer Research vol 70 no 11 pp4481ndash4489 2010
[13] H Li and M drsquoAnjou ldquoPharmacological significance of glyco-sylation in therapeutic proteinsrdquoCurrent Opinion in Biotechnol-ogy vol 20 no 6 pp 678ndash684 2009
[14] J Sharifi L A Khawli J L Hornick and A L EpsteinldquoImproving monoclonal antibody pharmacokinetics via chem-ical modificationrdquo The Quarterly Journal of Nuclear Medicinevol 42 no 4 pp 242ndash249 1998
[15] T Igawa H Tsunoda T Tachibana et al ldquoReduced eliminationof IgG antibodies by engineering the variable regionrdquo ProteinEngineering Design and Selection vol 23 no 5 pp 385ndash3922010
[16] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010
[17] J E Gessner H Heiken A Tamm and R E Schmidt ldquoThe IgGFc receptor familyrdquoAnnals of Hematology vol 76 no 6 pp 231ndash248 1998
[18] ICH ldquoICH Q9 quality risk managementrdquo in Proceedings ofthe International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse 2005
[19] D J Slamon G M Clark S G Wong W J Levin AUllrich andW L McGuire ldquoHuman breast cancer correlationof relapse and survival with amplification of the HER-2neuoncogenerdquo Science vol 235 no 4785 pp 177ndash182 1987
[20] D J Slamon W Godolphin L A Jones et al ldquoStudies ofthe HER-2neu proto-oncogene in human breast and ovariancancerrdquo Science vol 244 no 4905 pp 707ndash712 1989
[21] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005
[22] Z Mitri T Constantine and R OrsquoRegan ldquoThe HER2 recep-tor in breast cancer pathophysiology clinical use and newadvances in therapyrdquo Chemotherapy Research and Practice vol2012 Article ID 743193 7 pages 2012
[23] L F Flores-Ortiz V R Campos-Garcıa F C Perdomo-Abundez N O Perez and EMedina-Rivero ldquoPhysicochemicalproperties of Rituximabrdquo Journal of Liquid Chromatography ampRelated Technologies vol 37 no 10 pp 1438ndash1452 2014
[24] V Perez Medina Martınez M E Abad-Javier A J Romero-Dıaz et al ldquoComparability of a three-dimensional structure inbiopharmaceuticals using spectroscopic methodsrdquo Journal ofAnalytical Methods in Chemistry vol 2014 Article ID 95059811 pages 2014
[25] C E Espinosa-de la Garza F C Perdomo-Abundez J Padilla-Calderon et al ldquoAnalysis of recombinant monoclonal antibod-ies by capillary zone electrophoresisrdquoElectrophoresis vol 34 no8 pp 1133ndash1140 2013
[26] Beckman Coulter CE Separation of N-Linked OligosaccharidesReleased from Recombinant Monoclonal Antibody ApplicationInformation Beckman Coulter Pasadena Calif USA 2004
[27] M P Miranda-Hernandez C A Lopez-Morales N DRamırez-Ibanez et al ldquoAssessment of physicochemical prop-erties of rituximab related to its immunomodulatory activityrdquoJournal of Immunology Research In press
[28] J P Carter and L G Presta ldquoImmunoglobulin variantsrdquo USPatent 5821337 1998
[29] The United States Pharmacopeial Convention ldquoTrastuzumabrdquoMedicines compendium Version 10 2013
[30] S N Telikepalli O S Kumru C Kalonia et al ldquoStructuralcharacterization of IgG1 mAb aggregates and particles gener-ated under various stress conditionsrdquo Journal of PharmaceuticalSciences vol 103 no 3 pp 796ndash809 2014
[31] T Ishikawa N Kobayashi C Osawa E Sawa and K Waka-matsu ldquoPrevention of stirring-inducedmicroparticle formationin monoclonal antibody solutionsrdquo Biological and Pharmaceu-tical Bulletin vol 33 no 6 pp 1043ndash1046 2010
[32] A J Cordoba B-J Shyong D Breen and R J HarrisldquoNon-enzymatic hinge region fragmentation of antibodies insolutionrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 818 no 2 pp 115ndash1212005
[33] V Kayser N Chennamsetty V Voynov B Helk and B L TroutldquoTryptophan-tryptophan energy transfer and classification oftryptophan residues in proteins using a therapeuticmonoclonalantibody as a modelrdquo Journal of Fluorescence vol 21 no 1 pp275ndash288 2011
[34] P R Callis ldquo 1L119886
and 1L119887
transitions of tryptophan applicationsof theory and experimental observations to fluorescence ofproteinsrdquoMethods in Enzymology vol 278 pp 113ndash150 1997
[35] R W Cowgill ldquoFluorescence and the structure of proteins IIFluorescence of peptides containing tryptophan or tyrosinerdquoBiochimica et Biophysica Acta vol 75 pp 272ndash273 1963
[36] R W Cowgill ldquoFluorescence and the structure of proteins IEffects of substituents on the fluorescence of indole and phenolcompoundsrdquo Archives of Biochemistry and Biophysics vol 100no 1 pp 36ndash44 1963
[37] J Baselga and J Albanell ldquoMechanism of action of anti-HER2monoclonal antibodiesrdquoAnnals ofOncology vol 12 supplement1 pp S35ndashS41 2001
[38] J Baselga J AlbanellM AMolina and J Arribas ldquoMechanismof action of trastuzumab and scientific updaterdquo Seminars inOncology vol 28 no 5 supplement 16 pp 4ndash11 2001
