Mass Spectrometry for Characterization of Monoclonal Antibodies: Regulatory Considerations

Transcription

1 Mass Spectrometry for Characterization of Monoclonal Antibodies: Regulatory Considerations Jun Park, Ph.D. Division of Monoclonal Antibodies Office of Biotechnology Products OPS/CDER Food and Drug Administration 1

2 Disclaimer The content of the presentation may not necessarily reflect official policy at the FDA. 2

3 Outline Introduction Mass Spectrometry Protein Structure Higher-Order Structure (Conformation) Rapid Detection and Identification of Adventitious Agents using PCR & MS Conclusion Acknowledgement 3

4 Manufacturing of Monoclonal Antibodies: Regulatory Perspective The goal of manufacturing is to consistently provide (desired) product that reflects the materials used in clinical trials regarding safety and efficacy. (S. Kozlowski & P. Swann., Adv. Drug Del. Rev. 58:707, 2006) Critical Quality Attributes (ICH Q8R1): A physical, chemical, biological or microbiological properties or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. 4

5 Regulatory Considerations: How much of the desired product can we evaluate/control? Release tests (specifications) Characterization? Process (validation & control) From S. Kozlowski & P. Swann., Adv. Drug Del. Rev. 58:707,

6 Protein Structures/ Aggregates/ Particles Monomers 1 o, 2 o, & 3 o structures Post-translational modifications Size and charge variants Aggregates Particles Subvisible Particles Visible Particles nm m mm cm (10 7 nm) 10 Soluble Insoluble 6

7 Mass Spec-based techniques for structural characterization of monoclonal antibodies Zhang et al., Mass Spec. Rev., 28: ,

8 Protein structure determination by MS Mass determination Amino acid sequence Location of disulfide linkages Glycan structure and profiling Post-translational modifications Deamidation Oxidation Glycation Heavy chain C-terminal processing: C-terminal lysine N-terminal cyclization: pyroglutamic acid Fragmentation Conformation 8

9 Use of MS in Antibody Development Clone selection Cell culture Downstream purification Formulation development Stability studies Comparability studies Follow-on protein product characterization(?) 9

10 Characterization Workshop Questions With recent instrumentation improvements is the regulatory bar raised with respect to agency expectations as to complete profiling for minor chemical structures? Intact protein mass analysis is vastly improved in the past decade and LC- and CE-MS methods for intact proteins are revealing more and more information as to product related substances and impurities present. What level of these should be characterized? How do regulatory agencies view quantitative methods based on MS? 10

11 Quality Attributes of Monoclonal Antibody pyro-e D O O G D D O G O pyro-e D Pyro-Glu (2) Deamidation (3 x 2) Methionine Oxidation (2 x 2) D D Glycation (2 x 2) G G Glycans: High mannose, G0, G1, G1, G2 (5) K (9600) K Sialylation (5) C-terminal Lys (2) 2 x 6 x 4 x 4 x 5 x 5 x 2 =

12 Summary: Protein Characterization by MS Primary sequence: can be determined Variants: can be detected and identified, always not necessarily quantified (analytical power?) Glycoforms: can be mapped Stability monitoring including stressed samples Useful as an orthogonal technique Useful in combined approaches (e.g., enzyme/lc/ms) Quantitative applicability not well established 12

13 Analytical Methods: Protein Higher-Order Structures Secondary structure Far UV CD (helical structure) FTIR (beta-sheet) Raman Tertiary structure Near UV CD Second derivative UV Fluorescence (e.g., ANS) Raman Differential Scanning Calorimetry (DSC): thermal stability Free thiol analysis: disulfide bonds Mass Spectrometry (MS) - Ion Mobility MS - Hydrogen/Deuterium Exchange MS 13

14 Workshop Questions/Comments Comparability: Ion mobility MS and hydrogen deuterium exchange MS offer the potential to monitor and compare protein conformational attributes. Although their use is in its infancy, how are such methods viewed from a regulatory perspective? What type of data should be submitted in filings? Are there any expectations as to quantitative results or method reproducibility? Can these methods be used in comparing biosimilars with innovator products and if so to what extent? Are MS methods for assessing protein conformation viewed as supplementary to alternatives or even capable of replacing existing methods, or are there deficiencies in our ability to discern the meaning of the results? 14

15 Yijia Jiang, 2008 ACS Conference 15

16 IgG 2 Isoforms: Disulfide Shuffling IgG 2 IgG 1 IgG 2 Isoforms have different Conformational structures Binding activities Martinez et al., Biochemistry, 47: 7496, 2008 Thomas Dillon et al., JBC, 283: 16206,

17 CD Signal No detectable secondary structure change (~ 5%) as determined by Far UV CD and FTIR Cloudy Clear Wavelength (nm) Li Shi, 2009 ACS Conference 17

18 Higher-order structure vs. biological activity for complex molecules, the physicochemical information may be extensive but unable to confirm the higher-order structure which, however, can be inferred from the biological activity ICH Q6B (1999): Specifications for Biotechnology Products 18

19 Summary: Higher-Order Structure Can be assessed with stress & multiple techniques Does not substitute for functional assays Limited ability to detect micro-heterogeneity Most analytical methods for higher-order structures provide qualitative information Used as a characterization assay Need to develop reliable quantitative analytical techniques Ion mobility MS and hydrogen deuterium exchange MS: potential for protein conformations 19

20 Detection and Identification of Adventitious Agents by Mass Spectrometry Contaminations with adventitious agents (i.e., viruses, mycoplasma, bacteria, fungi) represent a potential critical risk for the manufacture of therapeutic proteins. Current practices for the detection of such adventitious agents rely on the culture of specimen that require weeks or even months of observation. Potential powerful approach for rapid detection and identification of such adventitious agents using polymerase chain reaction (PCR) and electrospray ionization mass spectrometry (ESI-MS). 20

21 Ranga Sampath, Ibis Biosciences, 2009 PDA Cell Substrate Workshop 21

22 Detection and Identification of Adventitious Agents using PCR & ESI-MS John Kolman, Bioreliance, 2009 PDA Cell Substrate Workshop 22

23 Conclusion Mass spectrometry has become an essential tool for the structural characterization of therapeutic proteins. Higher-order structure variation is still difficult to detect quantitatively. Mass spectrometry has (may offer) a promising means for: Protein conformational attributes: Ion mobility MS and hydrogen deuterium exchange MS Rapid detection and identification of unknown adventitious agents Can we completely characterize protein structure with biochemical methods? Do we truly know everything? How much change (specifications, comparability, follow on protein products) in a product is biologically meaningful or tolerable? 23

24 Acknowledgements Division of Monoclonal Antibodies / OBP/ CDER: Patrick Swann - Kurt Brorson Chana Fuchs - Carla Lankford Ruth Cordoba-Rodriguez - Ram Sihag Barbara Rellahan - Sarah Kennett Michele Dougherty - Rashimi Rawat Laurie Graham - Lixin Xu Authors of cited papers Presentations from Industry 24

25 To be added for future presentation Use mass spec for studying Higher-order structure by Ion Mobility MS or Hydrogen/Deuterium Exchange (HDX) MS IgG4 Fab-arm exchange with endogenous human IgG4 measured by MS and binding assay PK Host cell protein (see Water s talk) Other impurities 25