INTRODUCTIONAny manufacturer seeking to develop and market a biopharmaceutical product requires comprehensive physicochemical structural characterization of the (glyco)protein. For biosimilar products, this task is magnified and forms the basis for further comparability. In line with various regulatory guidelines (see below), characterization should be performed at distinct stages of development. Initially, batches of the target originator molecule should be studied to de-termine the exact sequence of the target protein, the post-translational modifications and the variability of quality attributes in batches over time. Then, once the biosimilar product is manufactured, characterization needs to be performed to confirm it’s structure. Finally, manufacturers must provide com-parative data for the biosimilar side-by-side with the originator molecule. Analyses must determine if proteins have the same biochemical, biophysical and physiological attributes. Analytical strategies should include a battery of physicochemical techniques for primary and higher order structure (Table1) with appropriate sensitivity to detect product related variants and impurities.
The complexities of biomanufacturing make exact replication of the originators molecule impossible. Instead, biosimilars may be granted approval on the basis of analytical, pre-clinical and clini-cal data which show they are highly similar to the reference product at 3 levels:
• Quality • Safety• Efficacy
Table 2 describes the common step-by-step approach to take in a biosimilar analytical development program.
BIOSIMILAR DEFINITIONS“A biosimilar is a copy version of an already authorized biological medicinal product (reference product) with demonstrated similarity in physicochemical characteristics, efficacy and safety, based on a comprehensive comparability exercise”1
COPY BIOLOGICS• “True” biosimilars – also called similar biotherapeutic products, follow-on protein products, subsequent entry protein products, subsequent entry biologics, similar biologics etc. depending
on regulatory region are compared to originator. Copy biologic products not developed and assessed in a scientific comparability program vs. a reference product are referred to as non-comparable biologics (NCB).
• Biobetters are an improvement of an existing drug by e.g pegylation. Treated as new molecule, not a biosimilar.
REGULATORY BACKGROUND
OBJECTIVE: STRUCTURAL CHARACTERIZATION OF BIOSIMILARSThe first step in development is the determination of the exact sequence of the originator protein. This involves de-novo MS/MS sequencing with careful interpretation and assignment of levels of post-translational modifications in multiple batches. The objective is to define the “Quality Target Product Profile (QTPP), representing the desired specifications for the final product. It is es-sential at this stage that the correct AA sequence is deduced. Figure 1 illustrates a case in which sequence differed from that anticipated from published data.
During initial cell-line development to produce the desired biosimilar, screening characterization can also be used to aid selection of appropriate clones, such as those expressing the desired glycosylation profile.
Once the biosimilar product has been manufactured, extensive side-by-side comparison is performed. There are many variables to consider such as:• choice of reference material – where it is manufactured• number of batches of biosimilar vs number of batches of originator• changes in originator product over time.
Some regulatory guidelines mention “fingerprinting” of quality attributes to establish biosimilarity. Figures 2- show examples of many of the techniques regularly used to generate these data in an antibody case study.
METHODS Analytical strategies include a battery of physicochemical techniques for primary and higher order structure with appropriate sensitivity to detect product related variants and impurities. Techniques include MALDI-TOF MS, ESI-MS, ESI-MS/MS, GC-MS, HPAEC-PAD, cIEF, CD and AUC which can be used to establish comparative analysis on glycoprotein products.
ANALYTICAL CHARACTERIZATION OF BIOSIMILAR PRODUCTS TO ESTABLISH BIOSIMILARITYDR. FIONA M GREER, GLOBAL DIRECTOR, BIOPHARMA SERVICES DEVELOPMENT, SGS M-SCAN
TABLE 1: WHAT REGULATIONS COVER PHYSICOCHEMICAL-CHARACTERIZATION?
CONCLUSIONS• AdvancesinMSinstrumentationandProteomic/GlycomicstrategiesenablerapididentificationofproteinproductsandtheirPostTranslationalModifications,includingglycosylation.MS
techniquesareapplicabletocharacterizationatallstagesofdevelopment,butessentialfordeterminationoforiginatorsequence.• MStechniquesalonearenotenoughforfullphysicochemicalcharacterizationandcomparisonofbiosimilarwithreferenceproductsforregulatorypurposes.Otherorthogonalmethods
shouldalsobeincluded.• Strategiesforcomparabilitymustincludeassessmentofbothprimaryandhigherorderstructure.Batch-to-batchvariationoftheproductshouldbedeterminedforboththebiosimilarand
theoriginator.Inessence,ananalyticalstrategywillfollowcloselytherequirementsoftheICHguidelineQ6B.• Thisextensivecomparabilityiscarriedoutpriortoanyclinicalassessmentofthebiosimilarproduct.
REFERENCES 1. Weise,Metal.,NatureBiotechnology29,690-693(2011).2. QualityConsiderationsinDemonstratingBiosimilaritytoaReferenceProteinProducthttp://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/
UCM291134.pdf.3. ScientificConsiderationsinDemonstratingBiosimilaritytoaReferenceProduct.http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291128.
pdf.4. Biosimilars:QuestionsandAnswersRegardingImplementationoftheBiologicsPriceCompetitionandInnovationActof2009.http://www.fda.gov/downloads/Drugs/GuidanceCompliance-
RegulatoryInformation/Guidances/UCM273001.pdf.5. GuidanceforIndustryFormalMeetingsBetweentheFDAandBiosimilarBiologicalProductSponsorsorApplicantshttp://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryIn-
formation/Guidances/UCM345649.pdf.6. EMAMultidisciplinary:Biosimilars.
http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000408.jsp&mid=WC0b01ac058002958c7. ICHHarmonisedTripartiteGuideline,TopicQ6B.Specifications:TestProceduresandAcceptanceCriteriaforBiotechnological/Biologicalproducts.Step4,ConsensusGuideline,March1999
(CPMP/ICH/365/96).
SAMPLE % α-HELIX % OTHER HELIX % β-SHEET % TURNS % ‘OTHER’
A 70 13 0 6 12
B 70 11 0 9 14
C 68 12 0 8 13
D 67 12 0 8 13
E 68 12 0 8 13
-10
-5
0
5
10
15
20
190 200 210 220 230 240 250 260
Wavelength (nm)
dE m
ol-1
dm
3 cm
-1
Sample ASample BSample CSample DSample E
Far-UVCirculardichroismanalysisofvariousbatchesoforiginatormoleculeandbiosimilar.
UNITED STATES
• BiologicsPriceCompetitionandInnovationAct(BPCIA)signedMarch2010.Newpathway-351(k)introducedintothePHSAct.
• Feb2012,FDAissueddraftGuidancedocu-ments(twoplusQ&A)toaccompanylegalacts,March2013,fourthguidanceissued2-5
EUROPE
• 2005,EMEAissuedguidelineson“Biosimi-lars”andcontinuestoissueandupdateincludingproductspecificguidances6
• ApprovedfirstBiosimilarin2006andnowhas14
REST OF WORLD
• Brazil,Australia,Turkey,India,Taiwan,Malaysia,Argentina,Mexico,Japan,Canada,SA.havesomeformofpathway.
• SomeadoptedEMAguides,otherswrotetheirown.
• Oct2009,WHO“GuidelineonEvaluationofSimilarBiotherapeuticProducts
TABLE 2: BIOSIMILAR ANALYTICAL DEVELOPMENT - A STEP-BY-STEP APPROACH
Inessence,ananalyticalstrategywillfollowtherequirementsofICHTopicQ6B7
“Specifications:TestProceduresandAcceptanceCriteriaforBiotechnological/BiologicalProducts”
FIGURE 1: ESTABLISHING THE QTPP
Duringinitialassessmentofatargetmolecule,peptidemassmappingwascarriedouttoob-tainconfirmationofexpectedstructure.However,aparticularpeptidestretchwasmissingfromtheMassMAP
ESQXXXXXXXRand ESQXXXXXXXRXXXXK
Acarefulsearchforexpectedy”ionswithinoneoftheMS/MSchannelswassuccessful.Howeverthemolecularweightoftheparentpeptidewas10amuhigherthananticipated.
Thefoundmasscorrespondingtothemissingpeptidewas+10amuhigher.FromMS/MSinterpretation,themassdifferencewasattributedtositeofthesecondresidue,possiblySerinetoProlinechange:
ESQXXXXXXXRXXXXKtoEPQXXXXXXXRXXXXK
FIGURE 2: CASE STUDY: ANTIBODY CHARACTERIZATION
Inthecaseofantibodies,thesizeandcomplexityofthemoleculerequiresLC/MS/MSapproaches.Thecomparabilityprogramincludes:
• MassspectrometryofintactproteinandreleasedL&Hchains
• AminoAcidCompositionAnalysis• N-terminalsequencing• Peptide“MAPPING”Analysis
(Sequencecoverage:100%LCand100%HC)
• Monosaccharideandsialicacidanalysis
• Oligosaccharidepopulationanalysis• SDS-PAGEanalysis• CircularDichroism• AnalyticalUltracentrifugation
FIGURE 3A: INTACT MASS MEASUREMENT FIGURE 3B: INTACT MASS MEASUREMENT
Mab +2 x G0F
Mab +1 x G0F+ 1 x G1F Mab +2 x G1F
Mab +1 x G1F+ 1 x G2F
G0FMassshift=+1444DaG1FMassshift=+162DaG2FMassshift=+324Da
Mab+2xG0F
Mab+1xG0F+1xG1F
Mab+2xG1F
Mab+1xG1F+1xG2F
IgG->N-Linkedbiantennarycorefucosylatedwithvaryingnumberofgalactoseresidues
FIGURE 4: MAPPING WORKFLOW FIGURE 5: ANALYSIS OF GLYCOSYLATION
FIGURE 6: COMPARABILITY ON BASIS OF CHARGE FIGURE 7: SECONDARY STRUCTURE ANALYSIS (FAR UV CD ANALYSIS 260-190NM)
ImagingcIEFcomparabilityassessmentof3batchesofintactMonoclonalAntibodyproducts
N-GlycansO-Glycans
IntactMassbyMALDIorESMSMonosaccharideCompositionAnalysis(LC&MS)
MonosaccharideCompositionGlycanPopulationScreeningGlycanAntennaryProfileGlycosylationSiteLinkageAnalysis
PermethylationMALDI,Nanospray-MS/MS&LinkageanalysisLC&MSmethods
Reduction Carboxymethylation
Specific Protease Digest
PNGase F
Sep-pak
Reductiveelimination
ORIGINATOR
STAGE 1
• RegulatoryConsultancyandAdvice
• FormulationDevelopment
• QualityControl,BatchConsistency,BatchRelease
• Microbiology,Mycoplasma,Mycobacteria,Sterility
• Virology(bulkharvest,bulkpurified,finalproduct)
• StabilityStorage(ICH)andTesting
• Process-RelatedImpurities
• Product-RelatedImpurities(HostCellProtein,HostCellDNA)
• ContainerTesting(extractables/leachables)
STAGE 2 STAGE 3 STAGE 4
SIDE-BY-SIDE COMPARISON
PRE & POST-COMPARABILITY
•Chemistrytestingofrawmaterials– Purity,identity&stability– Virusµbialdetection– Pre-MCBscreening– MCB,WCB,bulkcharacterization– EPC– Geneticstability
•Primaryandhigherorderstructure
•ICHQ6Banalyticalregime
•QualitativeandQuantitativeasses-smentofQualityAt-tributesofmultiplebatches
•Functional/potency: – In-vitro assays– Bioassays,ADCC– Cell-basedassays– Immunogenicityandbiomarkers
•Safety-Immunology-Virology
•Clinicaltrials•ComparativePK/PDstudiesinhealthyandtargetpopulations
•Clinicalefficacyinrandomizedparalleltrialsinsensitivepopulations
•RegulatoryAffairs•Bioanalysis
CELL LINE & PROCESS DEVELOPMENT
PHYSICOCHEMICAL BIOLOGICAL ACTIVITY PRE-CLINICAL CLINICAL COMPARISON & BIOANALYSIS
•DeterminationofExactSequence&Structure
•MS/MSde-novosequencing
•Protein/Glycoproteinsequencing
•DeterminationofPTMs
•QualityTargetPro-ductProfiledefined
T2144
STRUCTURAL CHARACTERIZATION AND CONFIRMATION
1. Aminoacidsequence
2. Aminoacidcomposition
3. Terminalaminoacidsequence
4. Peptidemap
5. Sulfhydrylgroup(s)anddisulfidebridges
6. Carbohydratestructure
PHYSICOCHEMICAL PROPERTIES
1. Molecularweightorsize
2. Isoformpattern
3. Extinctioncoefficient
4. Electrophoreticpattern
5. LiquidChromatographicpattern
6. Spectroscopicprofiles
Fuc
Asn - GlcNAc-GlcNAc- Man
Man – GlcNAc - Gal
Man - GlcNAc - Gal
