Antibody-Drug Conjugate Characterization and Quality Assurance

Transcription

1 Antibody-Drug Conjugate Characterization and Quality Assurance Sarah Kennett Division of Monoclonal Antibodies Office of Biotechnology Products OPS/CDER/FDA October 12,

2 Disclaimer The views and opinions expressed should not be used in place of regulations, published FDA guidances, or discussions with the Agency. 2

3 Overview Brief Introduction to Antibody-Drug Conjugates Current FDA Review Paradigm Expectations for Characterization/Specifications Unconjugated Antibody Cytotoxic Agent Antibody-Drug Conjugate 3

4 Immunoconjugates Whole mabs Fab F(ab )2 sfv Single VH domain mabs Cytotoxic Drug Radioisotope Cytokine Toxin (protein) Peptide Enzyme PEG Biotin/Streptavidin Other 4

5 Antibody-Drug Conjugates (ADCs) for the purposes of this talk Whole mabs Fab F(ab )2 sfv Single VH domain mabs Cytotoxic Drug Intent is to target a payload (the cytotoxic drug) to specific cells. 5

6 Commonly used Drugs and Linkers Doxorubicin based Calicheamicin based (Pfizer) Maytansinoid based (ImmunoGen) Auristatin based (Seattle Genetics) 6 Carter and Senter. Cancer J. 14:

7 Conjugation Examples 7 Nature Biotechnology. 26:

8 Conjugation Examples Fred Jacobson (Genentech) through Jun Park 8

9 Mechanism of Action Carter and Senter. Cancer J. 14:

10 There are Currently Numerous ADCs under IND From a 2010 review 10 Alley, et al. Current Opinion in Chemical Biology. 14:

11 How Review Works at FDA (CDER) Assigned to a Clinical Division in the Office of New Drugs based on indication (also Office of Clinical Pharmacology and many other Offices involved later in development) Product Quality (Office of Pharmaceutical Science) Primary reviewers from both OBP and ONDQA alternate lead (Office of Biotechnology Products and Office of New Drug Quality Assessment) Responsibilities: Unconjugated Antibody Drug, Linker, Drug-Linker Antibody-drug conjugate OBP (DMA) ONDQA OBP and ONDQA Currently Antibody-drug conjugates are classified as BLA products (Patient Protection and Affordable Care Act) 11

12 FDA Guidance on Immunoconjugates In addition to recommendations for unconjugated (naked) mab, manufacturers of immunoconjugates should address the following: a. Construction of the immunoconjugate. A full description of the reagents and the process used to construct an immunoconjugate should be submitted, including: A description of components including: the source, structure, production, purity and characterization A description of chemical components, such as linkers and chelating agents These should include determinations of residual impurities from synthesis or purification. The average ratio of coupled material to antibody and the number of conjugated moieties per antibody should be determined as the first step in establishing lot release criteria for the final product The stability of recombinant immunoconjugates should be studied carefully... b. Purity of the Immunoconjugate The amount of free antibody and free components in the final product should be determined with limits set for each. c. Immunoreactivity, Potency and Stability of the Immunoconjugate. Coupling of toxin or drug to an antibody may alter the activity of either component. Immunoreactivity before and after coupling should be assessed using appropriate methodology Activity of the non-immunoglobulin component of immunoconjugates, should be assessed by a potency assay whenever appropriate Limits on the percent change in immunoreactivity resulting from construction of the immunoconjugate should be established as part of product specifications. The immunoconjugate should be tested for stability in vitro (pooled human serum or plasma) Excerpts from Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use12 (US FDA,1997)

13 Unconjugated antibody The expectations are the same whether the antibody will be developed on its own or as part of an ADC. Characterization should include: Primary Structure (e.g., Peptide map/n-terminal sequencing, Q-TOF MS, free sulfhydryl) Secondary/Tertiary Structure (e.g., Disulfide bond- non-reduced MS, CD, FTIR, DSC) Size Variants - Aggregates/Fragments (e.g., r/nrce-sds, r/nrsds-page, SEC, AUC) Charge Variants (e.g., cief, icief, IEF, CEX) Glycosylation (e.g., oligosaccharide profile, monosaccharide analysis, sialic acid content) Other Post-translational Modification Antigen Binding Biological Activity as Appropriate (Potency assay and ADCC/CDC even when not MOA) Process Related Impurities (DNA, HCP, Protein A, recombinant proteins in culture media, adventitious agents, selection agents, growth enhancers, etc.) The amount of characterization required is dependent on the stage of development and the molecule. 13

14 Specifications Unconjugated antibody Identity Size Variants- Both Aggregate and Fragment (e.g., r/nrce-sds, r/nr-sds-page, SEC) Charge Variants (e.g. cief, icief, IEF, CEX) Potency (binding and bioassay, as appropriate) Glycosylation (antibody-dependent) Other modifications (antibody-dependent) Total protein Safety Bioburden, Endotoxin Process Related Impurities- Host Cell DNA, Host Cell Protein, Protein A, recombinant proteins in culture media, selection agents, growth enhancers, etc. Physical Characteristics Appearance, ph, etc. 14

15 Unconjugated antibody Expectations for comparability of unconjugated antibodies are the same whether the antibody will be developed on its own or as part of an ADC. Expectations may change as we become aware of newly understood properties of antibodies. IgG4 Fab arm exchange IgG2 disulfide scrambling Glycosylation knowledge (e.g., fucosylation effect on ADCC, sialylation anti-inflammatory properties) Expectations for engineered antibodies (e.g., glycoengineering or other mutations to reduce/enhance effector function) include demonstration that the engineered antibody has the expected (lack of) property. 15

16 Cytotoxic agent The expectations are the same whether or not the cytotoxic drug will be developed on its own or as part of an ADC. Examples of expected characterization and release testing (a combination of appropriate methods would be expected) Elemental analysis NMR MS/ICP-MS FTIR HPLC- purity (related substances) and assay CE? Optical rotation Residual solvents ICH Q3C Residue on ignition Melting range Appearance, moisture Characterization should allow for an assessment of the linkage of quality attributes across batches from toxicology and clinical lots to commercial lots. Comparability generally refers to comparability of the impurity profile, since identity, potency, and strength should not change. Comparable drug loading needs to be demonstrated. 16

17 Antibody drug conjugate Characterization includes the same characterization as is performed for the unconjugated antibody with a comparison to the unconjugated antibody results, as appropriate. Additional characterization includes an assessment of the conjugation. 17

18 Conjugation Assessment Fred Jacobson (Genentech) through Jun Park 18

19 Drug:Antibody Ratio versus Drug:Antibody Distribution 4:1 4: Percent Percent Drug:Antibody Drug:Antibody 19

20 Inter-chain disulfide conjugation Drug load potential variants 20

21 Purity/Potency Drug:Antibody Drug:Antibody Light/Heavy chain L0 L1 H0 H1 H2 H3 HIC RP-HPLC 21

22 Inter-chain disulfide conjugation Drug load potential variants HIC RP-HPLC 22

23 Antibody drug conjugate Characterization includes the same characterization as is performed for the unconjugated antibody with a comparison to the unconjugated antibody results, as appropriate. Additional characterization includes an assessment of the conjugation. Peptide maps for identification of sites of conjugation are easier to interpret for cys-linked drugs than for lys-linked drugs Can t always use same assays as for unconjugated antibodies Charged based assays for lysine-linked ADC or ADC with charged drug/linker SDS and MS based assays for cysteine-linked ADCs have some issues Lysine conjugation reaction also leads to some bonding with tyrosine. This is a less stable ester bond that is formed at low concentration and leads to release of free drug over time, and this would need to be controlled Need to understand what is stability indicating and include in stability protocols 23

24 Antibody drug conjugate Specifications Identity (dependent on attributes of conjugate and if other conjugates are manufactured at het same facility) Purity Size variants (r/nrsds-page, r/nrce-sds, SEC) Charge variants (IEF, cief, icief) Drug related impurities (RP-HPLC) Potency CE? Binding assay (if it has been demonstrated that conjugation does not affect binding, that conjugation process is well controlled, and that bioassay is robust and stability-indicating, there is potential for removal of this assay (DP)) Cytotoxicity Bioassay Purity/Potency Drug:antibody ratio (HIC, RP-HPLC) Drug:antibody distribution (HIC, RP-HPLC; including free antibody) Free Drug Total protein Safety Sterility, Endotoxin Physical Characteristics Appearance, Osmolality, ph, Polysorbate, Particulates (DP) CE? 24