application of high resolution pharmacokinetic techniques for · : geometric means (% cv) t 1/2,...

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

3

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


Mechanistic PK Model: Factors Modulating Pharmacokinetics of mAbs

4

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


Designing PK Studies for Biosimilars

5

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)


Conventional Approach to Comparative PK for mAbBiosimilar

6

Time

Concen

trationTime concentration curves

Bioequivalence testing of PK parameters(Cmax, Tmax, AUC or partial AUC)

Reference

Biosimilar


Conventional Approach to Comparative PK for mAbBiosimilar

7

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


High Resolution Approach for Comparative PK for mAbBiosimilars

8

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


Goal

9

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)


LC‐MS/MS Sample Processing

10

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


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

11 © 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 Biological Test System

12

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



Study analysis Limited power due to experimental design

Can be adequately powered Can be adequately powered


Selection of Immunocapture Antibody (High Abundance Glycopeptide ‐ G0)

13

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)


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)


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


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)


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


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


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


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 Embraces ComplexityThree Critical Components to Deliver Medicines

21

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


application of high resolution pharmacokinetic techniques for · : geometric means (% cv) t 1/2,...

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