Application of High Resolution Pharmacokinetic Techniques for Biosimilar DevelopmentWCBP ‐ “New and Emerging Analytical Technologies for Biotherapeutics” Plenary Session; Jan. 28, 2015
Tanmoy Ganguly, Ph.D.Senior Director, Research
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Executive Summary
• Biosimilar development is an exercise in the “science of comparison”• Comparative PK studies for biosimilar molecule should therefore provide the
necessary level of resolution to establish similarity to the Reference Protein Product (RPP)
• mAbs (and fusion proteins) are complex heterogeneous mixtures of multiple glycoforms• Comparative PK studies for biosimilar molecule should therefore not rely on
“aggregate” PK parameters to the RPP, but compare PK parameter s of individual glycoforms present in the biosimilar and RPP
• LC‐MS/MS based methods can be used to compare the PK parameters of multiple glycoforms• A robust method suitable for analysis in multiple matrices was developed that
provides PK parameters of multiple glycoforms and potentially can be used as platform for biosimilar development
2 © Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Pharmacokinetics of mAb Therapeutics
• Absorption• Mainly via the lymphatic system – SC or IM route• Typically slow ‐ Tmax ~ 2‐8 days• Bioavailability ‐ 50‐100%
• Distribution• Convective transport – blood to interstitial fluid to lymph
• Rate of fluid movement from blood to tissue and lymphatics• Sieving by vascular endothelium
• Low distribution in brain – tight junctions• Leaky in spleen, liver, and bone marrow
• Elimination• Majority by intracellular catabolism in tissues
• Fluid phase endocytosis• Recycling via FcRn ‐ saturable• mAb‐Anti‐Drug Antibody (ADA) complexes ‐may increase or decrease PK
• Receptor mediated endocytosis• Target mediated drug disposition (TMDD)• mAb‐FcRs complex
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Pharmacokinetics of mAb therapeutics are different and more complex than that of small moleculesPharmacokinetics of mAb therapeutics are different and more complex than that of small molecules
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Mechanistic PK Model: Factors Modulating Pharmacokinetics of mAbs
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kel_Ab-ADA
+ Target Ab-TargetKD_TNF
ksyn_TNF
kel_TNF
PLASMA
FcR Ab-FcR+KD_FcR
ksyn_FcR
kel_FcR
FcRn Ab-FcRn+KD_FcRn
ksyn_FcRn
kel_FcRn
ENDOSOME
TISSUE (VASCULAR + INTERSTITIAL)
kel_Ab-FcRADCC
kel_Ab-TNF clearance;TMDD
VP
VT
VE
Ab
kPT kTP
kel_Ab,Elysosomedegradation
kel_Ab,Premainingclearance
Ab
kTE
krecyc
ADAmax Dose Dose + Km
ADA
ADA
kT_ADA
ADA Dose
kel_Ab-C1qCDC
SC Dose
Ab
kabs_Ab,SC
Ab-ADA+KD_ADA
kel_ADA
Ab
C1q Ab-C1q+KD_C1q
ksyn_C1q
kel_C1q
Ab
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Designing PK Studies for Biosimilars
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An appropriate combination of test systems, experimental design and measurements is required to provide the necessary resolution for comparison of PK parametersAn appropriate combination of test systems, experimental design and measurements is required to provide the necessary resolution for comparison of PK parameters
TEST SYSTEMS
Preclinical species
(rodent, rabbit primate)
Human (NHV, patients)
EXPERIMENTAL DESIGN
Dosing regimen
Route
Sampling times
MEASUREMENTS
A) Free drug (ELISA)
B) Specific glycoforms (LC‐MS/MS)
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Conventional Approach to Comparative PK for mAbBiosimilar
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Time
Concen
trationTime concentration curves
Bioequivalence testing of PK parameters(Cmax, Tmax, AUC or partial AUC)
Reference
Biosimilar
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Conventional Approach to Comparative PK for mAbBiosimilar
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Method: Male Balb/C mice (12/group) were dosed with 30 mg/kg test articles using SC route and serum drug concentration was measured at 13 sample points post dosing using ELISA. PK parameters and bioequivalence testing done using Non‐Compartmental Analysis module of Phoenix WinNonlin®
• No statistically significant (p<0.05) differences observed for the above pharmacokinetic parameters• Biosimilar is equivalent to Reference based on Cmax and partial AUC PK parameters• No statistically significant (p<0.05) differences observed for the above pharmacokinetic parameters• Biosimilar is equivalent to Reference based on Cmax and partial AUC PK parameters
Test Article Cmax(mg/ml)
AUCinf(mg*d/ml)
AUC(0‐14)(mg*d/ml) t1/2 (day) Tmax (day)
CL (ml/d/kg)
Vd(ml/kg)
Reference 317.7 (10.0) 3023 (20.5) 2477 (9.5) 4.6 ± 2.5 1 [1, 2] 10.1 ± 1.8 61.9 ± 20.5
Biosimilar 289.9 (9.3) 2632 (26.9) 2176 (14.4) 4.7 ± 2.6 2 [1, 2] 11.7 ± 3.0 71.0 ± 20.9
Cmax, AUCinf and AUC(0‐14) : Geometric means (% CV) t1/2, CL, and Vd : Arithmetic means ± SD Tmax: Median [min, max]
Test dependent Units Ref Article RefLSM Test Article TestLSM Ratio_%Ref_ CI_90_Lower CI_90_Upper Power
Ln (Cmax) ng/mL Reference 12.67 Biosimilar 12.58 91.2 85.06 97.84 0.9996
Ln(AUC0‐14) day*ng/mL Reference 14.72 Biosimilar 14.61 87.8 80.10 96.33 0.9889
Time (Day)
Dru
g se
rum
con
cent
ratio
n (n
g/m
l)M
ean
SD
0 10 20 30 400
100000
200000
300000
400000
ReferenceBiosimilar
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
High Resolution Approach for Comparative PK for mAbBiosimilars
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Time
Concen
tration
Population“less resolved”
N‐glycosylated at Asn‐297
Conventional Approach Time
Concen
tration
Individual glycoforms“more resolved”
Hundreds of possible glycoforms
High Resolution Approach
Versus
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Goal
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Develop a method to compare PK parameters of glycoforms that is suitable for biosimilar development
‐ Provides more resolution (discrimination) versus ELISA method‐ Utilizes the resolving power of LC and detection of mass spectroscopy‐Works in multiple matrices (rodent, NHP and human plasma)‐ Used as a platform for biosimilar mAbs (and fusion proteins)
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
LC‐MS/MS Sample Processing
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Sample Preparation
•Capture mAb in serum (rodent, NHP or human) using suitable capture antibody immobilized on column/beads
•Wash column and elute mAb•Reduce and alkylate the isolated mAb
Sample Processing
•Enzymatic digestion of the isolated mAb•Spike suitable peptide standards
Sample Analysis
•LC‐MS/MS of the glycopeptides•Resolution of the LC•MS signal intensity
•Data analysis
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Considerations for Methods Development: Factors Dependent on the Biologic
• Type of biologic • mAb or fusion protein
• Number of Glycosylation sites • Single glycosylation site in mAb in Fc region• Multiple glycosylation sites in protein and in Fc
• Type of Glycosylation • N or O linked glycosylation
• Number of Glycosylation sites detected by the MRM method• Single glycosylation site in mAb in Fc region• Multiple glycosylation sites in fusion protein
• LC method used• Abundance of glycopeptide(s)• Resolution of between glycopeptide(s)
• Lower abundance can be quantified if well resolved
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Considerations for Methods Development: Factors Dependent on the Biological Test System
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Parameter Rodent (mouse) NHP (Cynomolgus monkey) Human (NHV or patient population)
Experimental design Higher than therapeutic dosing
Higher than therapeutic dosing
Limited to therapeutic dose
Limited ability for temporal sampling ‐multiple animals for individual time points
Temporal bleeding in same animal
Temporal bleeding in same subject
Sample prep Smaller volumes Higher volume Higher volume
Enrichment can be done with anti‐human or anti‐Fc capture Ab with limited interference
Enrichment requiresdevelopment of anti‐idiotypic/binding capture Abs (polyclonal or monoclonal)
Enrichment requiresdevelopment of anti‐idiotypic/binding capture Abs (polyclonal or monoclonal)
Usually limited interference from capture Ab in matrix
Usually interference from capture Ab observed (double affinity purification) in matrix
Usually interference from capture Ab observed (double affinity purification) in matrix
Experimental conditions for specific glycoforms have to be optimized
Experimental conditions for specific glycoforms have to be optimized
Experimental conditions for specific glycoforms have to be optimized
Study analysis Limited power due to experimental design
Can be adequately powered Can be adequately powered
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Selection of Immunocapture Antibody (High Abundance Glycopeptide ‐ G0)
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Several product specific monoclonal antibodies (AB1‐ AB5) as well as double affinity purified polyclonal anti‐idiotypic(poly‐AB) were screened for immunocapture of mAb from monkey or human serum. Resolution of the resultant glycopeptides was tested by LC‐MS/MS under experimental conditions
Time (min)
Intensity G0
AB3
AB5
AB4
Poly‐AB
(Human Serum)(Cynomolgus Serum)
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Selection of Immunocapture Antibody (Low Abundance Glycopeptide ‐ HM7)
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• Immunocapture with Ab3 and Ab4 showed the cleanest MS/MS spectra in both NHP and human serum for high and low abundance glycopeptides
• poly Ab showed cleanest spectra in human serum but not suitable for NHP serum due to cross reactivity with NHP IgGs.
• Immunocapture with Ab3 and Ab4 showed the cleanest MS/MS spectra in both NHP and human serum for high and low abundance glycopeptides
• poly Ab showed cleanest spectra in human serum but not suitable for NHP serum due to cross reactivity with NHP IgGs.
Time (min)
Intensity HM7
(Human Serum)(Cynomolgus Serum)
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Linearity of Surrogate Marker Peptide and Glycopeptides
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Large number of peptides were screened to identify a peptide to serve as a surrogate for the intact mAb; linearity was then determined. Similarly linearity was determined for high and low abundance glycans based on maximal and minimal expected serum concentrations
Linearity was established for marker peptide, high (G1F) and low (HM5) abundance glycopeptides under experimental conditionsLinearity was established for marker peptide, high (G1F) and low (HM5) abundance glycopeptides under experimental conditions
y = 0.1806x + 0.01R² = 0.9999
012345678
0 10 20 30 40 50Spiked mAb (μg
M Pep
/ (M
Pep
*)
y = 0.0826x ‐ 0.0438R² = 0.9911
0
0.5
1
1.5
2
2.5
3
3.5
4
0 10 20 30 40 50Spiked mAb (μg
G1F / (M
Pep
*)y = 0.0007x ‐ 0.0005
R² = 0.9957
0
0.005
0.01
0.015
0.02
0.025
0.03
0 10 20 30 40 50Spiked mAb (μg
HM5 / (M Pep
*)
M Pep = Marker Peptide used as surrogate for intact mAb
Backbone marker peptide
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
ELISA versus LC‐MS/MS Method
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Concentration‐time curves measured by high resolution method (LC‐MS/MS) are comparable to those measured using ELISA methodConcentration‐time curves measured by high resolution method (LC‐MS/MS) are comparable to those measured using ELISA method
PK of mAb in cynomolgus monkey (3/group), surrogate maker peptide for intact molecule was measured by LC‐MS/MS; serum free drug concentration was measured using ELISA
Time (Day)
Seru
m c
once
ntra
tion(
g/m
L)M
ean
SD
0 10 20 30 400
50
100
150
200
250
300
350
Reference (ELISA)Reference (LC-MS/MS)
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
High Resolution PK in Mice
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PK profiles can be generated for all of the dominant glycoforms and large numbers of minor species. Similar profiles indicate similar function across multiple interactions.
• Animals were treated with a single subcutaneous dose of reference or test product
• Serum levels of 17 glycopeptides were assessed over 42 days
• Representative comparative data is shown for one (HM5) of the 17 tested glycopeptides
• In the comparative study, variance among 4 animals per group was modest through the 42 day time point, allowing for a sensitive comparison
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Goal – Higher resolution than conventional (ELISA ) PK study
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Equivalent “intact” molecule PK parameters do not translate to equivalent glycoform PK parameters Equivalent “intact” molecule PK parameters do not translate to equivalent glycoform PK parameters
PK of two mAbs differing in N‐glycosylation were compared in Balb/C mice (4/group/time point) under identical conditions and serum free drug concentration was measured using ELISA; specific glycoforms (17 glycopeptides) were measured by LC‐MS/MS.
Intact molecule
Time (Day)
Seru
m c
once
ntra
tion
(ng/
mL)
Mea
n S
D
0 10 20 30 400
100000
200000
300000
mAb1mAb2
G2F-NeuAc
Time (Day)
Seru
m c
once
ntra
tion
(ng/
mL)
Mea
n S
D0 10 20 30 40 50
0
50000
100000
150000
200000
mAb1mAb2
HM5
Time (Day)
Seru
m c
once
ntra
tion
(ng/
mL)
Mea
n S
D
0 10 20 30 40 500
500
1000
1500
2000
2500
mAb2mAb1
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Conclusions
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• A robust LC‐MS/MS method was developed to detect multiple glycoforms of mAb:
• PK parameters showed orthogonality with ELISA method based on monitoring with a unique marker peptide
• Works in multiple matrices with appropriate method modifications in the Immunocapture step and LC method
• Provides the necessary resolution and discriminatory ability (relative to “aggregate “PK by ELISA)
• Amenable to other mAbs and can be used as a platform
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Acknowledgements
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Ganlin Zhao – Study design and NCA AnalysisYanjie Jiang – Analytical Nancy Dussault – In vivo dosing and samplingRyan Nolan – Mechanistic PK modeling
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.
Momenta Embraces ComplexityThree Critical Components to Deliver Medicines
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Understanding the nonlinear chemical and biosynthetic
reactions that drive production
Control of Manufacturing
High resolution physicochemical analytics platform to thoroughly
characterize any product
Thorough Structural Characterization
Thorough Biological Characterization
High resolution biology applied pre‐clinically and in clinical settings
• Generic Lovenox®• Generic Copaxone®(under FDA review)
Biosimilars • Necuparanib oncology Phase 1/2
• 3 novel autoimmune drugs preparing for the clinic in 2016:
• hsIVIg• SIF3• Anti‐FcRn
Novel Drugs
ANDA Generics• 2 programs elected by
Baxter:• M923 (Humira®)• M834
• Additional candidates in development
© Momenta Pharmaceuticals 2015. May not be reproduced or further distributed, in whole or in part, without Momenta’s prior approval.