Application of High Resolution Pharmacokinetic Techniques for Biosimilar Development

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1 Application of High Resolution Pharmacokinetic Techniques for Biosimilar Development WCBP New and Emerging Analytical Technologies for Biotherapeutics Plenary Session; Jan. 28, 2015 Tanmoy Ganguly, Ph.D. Senior Director, Research

2 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

3 Pharmacokinetics of mab Therapeutics Absorption Mainly via the lymphatic system SC or IM route Typically slow Tmax ~ 2 8 days Bioavailability % 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 Fc Rs complex Pharmacokinetics of mab therapeutics are different and more complex than that of small molecules 3

4 Mechanistic PK Model: Factors Modulating Pharmacokinetics of mabs ADA max Dose Dose + K m ADA Dose ADA SC Dose Ab + ADA k T_ADA K D_ADA Ab-ADA PLASMA k el_ab-ada k abs_ab,sc k el_ada remaining clearance k el_ab,p Ab k syn_tnf + Target K D_TNF Ab-Target k el_tnf k el_ab-tnf clearance; TMDD k syn_c1q Ab + C1q K D_C1q Ab-C1q k el_ab-c1q CDC k el_c1q V P k PT k TP TISSUE (VASCULAR + INTERSTITIAL) k syn_fc R Ab + Fc R K D_Fc R Ab-Fc R k el_ab-fc R ADCC k el_fc R k recyc V T k TE k syn_fcrn ENDOSOME lysosome degradation k el_ab,e Ab + FcRn K D_FcRn Ab-FcRn k el_fcrn V E 4

5 Designing PK Studies for Biosimilars TEST SYSTEMS Preclinical species (rodent, rabbit primate) Human (NHV, patients) EXPERIMENTAL DESIGN Dosing regimen Route Sampling times A) Free drug (ELISA) B) Specific glycoforms (LC MS/MS) MEASUREMENTS An appropriate combination of test systems, experimental design and measurements is required to provide the necessary resolution for comparison of PK parameters 5

6 Conventional Approach to Comparative PK for mab Biosimilar Reference Concentration Time Time concentration curves Biosimilar Bioequivalence testing of PK parameters (Cmax, Tmax, AUC or partial AUC) 6

7 Conventional Approach to Comparative PK for mab Biosimilar 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 Drug serum concentration (ng/ml) Mean SD Reference Biosimilar Time (Day) C max, AUC inf and AUC (0 14) : Geometric means (% CV) t 1/2, CL, and V d : Arithmetic means ± SD T max : Median [min, max] Test Article C max (mg/ml) AUC inf (mg*d/ml) AUC (0 14) (mg*d/ml) t 1/2 (day) T max (day) CL (ml/d/kg) V d (ml/kg) Reference (10.0) 3023 (20.5) 2477 (9.5) 4.6 ± [1, 2] 10.1 ± ± 20.5 Biosimilar (9.3) 2632 (26.9) 2176 (14.4) 4.7 ± [1, 2] 11.7 ± ± 20.9 Test dependent Units Ref Article RefLSM Test Article TestLSM Ratio_%Ref_ CI_90_Lower CI_90_Upper Power Ln (Cmax) ng/ml Reference Biosimilar Ln(AUC 0 14 ) day*ng/ml Reference Biosimilar 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

8 High Resolution Approach for Comparative PK for mab Biosimilars N glycosylated at Asn 297 Hundreds of possible glycoforms Versus Concentration Population less resolved Concentration Individual glycoforms more resolved Time Conventional Approach Time High Resolution Approach 8

9 Goal 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) 9

10 LC MS/MS Sample Processing 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 10

11 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

12 Considerations for Methods Development: Factors Dependent on the Biological Test System Parameter Rodent (mouse) NHP (Cynomolgus monkey) Human (NHV or patient population) Experimental design Higher than therapeutic dosing Limited ability for temporal sampling multiple animals for individual time points Higher than therapeutic dosing Temporal bleeding in same animal Limited to therapeutic dose Temporal bleeding in same subject Sample prep Smaller volumes Higher volume Higher volume 12 Study analysis Enrichment can be done with anti human or anti Fc capture Ab with limited interference Usually limited interference from capture Ab in matrix Experimental conditions for specific glycoforms have to be optimized Limited power due to experimental design Enrichment requires development of antiidiotypic/binding capture Abs (polyclonal or monoclonal) Usually interference from capture Ab observed (double affinity purification) in matrix Experimental conditions for specific glycoforms have to be optimized Can be adequately powered Enrichment requires development of antiidiotypic/binding capture Abs (polyclonal or monoclonal) Usually interference from capture Ab observed (double affinity purification) in matrix Experimental conditions for specific glycoforms have to be optimized Can be adequately powered

13 Selection of Immunocapture Antibody (High Abundance Glycopeptide G0) 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 (Cynomolgus Serum) Intensity G0 Time (min) (Human Serum) AB3 AB4 AB5 Poly AB 13

14 Selection of Immunocapture Antibody (Low Abundance Glycopeptide HM7) (Cynomolgus Serum) Intensity HM7 Time (min) (Human Serum) 14 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.

15 Linearity of Surrogate Marker Peptide and Glycopeptides 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 M Pep / (M Pep*) Backbone marker peptide y = x R² = Spiked mab (μg G1F / (M Pep*) y = x R² = Spiked mab (μg HM5 / (M Pep*) y = x R² = Spiked mab (μg M Pep = Marker Peptide used as surrogate for intact mab Linearity was established for marker peptide, high (G1F) and low (HM5) abundance glycopeptides under experimental conditions 15

16 ELISA versus LC MS/MS 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 350 Serum concentration( g/ml) Mean SD Reference (LC-MS/MS) Reference (ELISA) Time (Day) Concentration time curves measured by high resolution method (LC MS/MS) are comparable to those measured using ELISA method 16

17 High Resolution PK in Mice 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 17

18 Goal Higher resolution than conventional (ELISA ) PK study 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. Serum concentration (ng/ml) Mean SD Intact molecule mab1 mab Time (Day) Serum concentration (ng/ml) Mean SD Serum concentration (ng/ml) Mean SD HM Time (Day) G2F-NeuAc mab1 mab2 mab1 mab Time (Day) Equivalent intact molecule PK parameters do not translate to equivalent glycoform PK parameters 18

19 Conclusions 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 19

20 Acknowledgements Ganlin Zhao Study design and NCA Analysis Yanjie Jiang Analytical Nancy Dussault In vivo dosing and sampling Ryan Nolan Mechanistic PK modeling 20

21 Momenta Embraces Complexity Three Critical Components to Deliver Medicines Thorough Structural Characterization Control of Manufacturing Thorough Biological Characterization High resolution physicochemical analytics platform to thoroughly characterize any product Understanding the nonlinear chemical and biosynthetic reactions that drive production High resolution biology applied pre clinically and in clinical settings ANDA Generics Generic Lovenox Generic Copaxone (under FDA review) Biosimilars 2 programs elected by Baxter: M923 (Humira ) M834 Additional candidates in development Novel Drugs Necuparanib oncology Phase 1/2 3 novel autoimmune drugs preparing for the clinic in 2016: hsivig SIF3 Anti FcRn 21