Application of a Mass Spectrometry Based Multi-Attribute-Method (MAM) for QC Release and Stability Testing of Protein Therapeutics

Transcription

1 Application of a Mass Spectrometry Based Multi-Attribute-Method (MAM) for QC Release and Stability Testing of Protein Therapeutics Tamer Eris, Principal Scientist Process Development Amgen, Thousand Oaks, CA CASSS CMC Strategy Forum July 18 th, 2016

2 Key Drivers for a Multi Attribute Method (MAM) Attribute focused Process Development and Quality Control MAM allows for direct monitoring of relevant product quality attributes rather than indirect measurement by conventional methods MAM has capability for new peak detection Reduce Cost of Development and Quality Control MAM has potential for reduced number of assays for process development, product disposition, and improving cycle time Potential for modality independent, platform based method Quality-By-Design (QBD) Application of MAM method to process development and characterization embraces QBD principles For Internal Use Only. Amgen Confidential. 2

3 Identification and Selection of the Critical Quality Attributes is Key for Application of MAM Target Product Profile Product Quality Attribute Assessments Quality Target Product Profile (QTPP) Indication & use Dosage & administration Tolerability Dosage forms & strength Efficacy Safety/side effects Value & access Attribute definition Product quality attribute assessment Potential impact for safety/efficacy Critical quality attribute selection Attribute range determination Attribute focused molecule & process design & development For Internal Use Only. Amgen Confidential. 3

4 The QTPP guides product quality characteristics prospectively Definition: A prospective summary of the quality characteristics of a drug product that ideally will be achieved to ensure the desired quality, taking into account safety and efficacy of the drug product The quality target product profile forms the basis of design for the development of the product. Considerations for the Quality Target Product Profile (QTPP) could include: Intended Use in Clinical Setting, Route of Administration, Dosage Form, Delivery Systems Dosage Strength(s) Container Closure System Therapeutic Moiety Release or Delivery and Attributes affecting Pharmacokinetic Characteristics Appropriate to the Drug Product Dosage Form being Developed Drug Product Quality Criteria (e.g. sterility, purity, stability, and drug release) Appropriate for the Intended Marketed Product Drug Substance and Dug Product are in Scope of QTPP

5 QTPP: PQA Ranges can be designed into the Product during Development Category DS Attributes Target Range Ranges Achieved Strength Concentration mg/ml mg/ml Asp Isomerization 2% % Trp Oxidation 5% 0.1% Met Oxidation 5% % Met Oxidation 5% 0.4% Met Oxidation 1% 7% % Met Oxidation 5% % High Mannose Glycans 2% 12% % Quality Protein Dimer/Oligomers (SEC HMW) 1% % Protein Fragmentation (rce LMW+MMW) 1% < 0.6% Glycation (LC K) 5% % Hydroxylysine (HC K) 2% < 0.1% Hydroxylysine (HC K) 4% % Osmolality mosm/kg mosm/kg Polysorbate % 0.015% % ph Host Cell Protein 100 ppm ppm Safety Residual Protein A < 6 ppm < 1 ppm Endotoxin 0.25 EU/mg EU/mg Bioburden 10 CFU/10 ml 0 5

6 MAM is a Peptide Map based on Mass Spectrometry that measures several Attributes in a single Assay Protein Sample Denature Proteins Denature the sample Reduce and alkylate Desalt Digest with trypsin Inject the digest LC/MS analysis Total Ion Chromatogram Extracted Ion Chromatograms 6

7 Quantification of Targeted attribute using Conventional Peptide Map with UV Detection Quantitation of modification of a peptide in CDR of the monoclonal antibody by reduced and alkylated Lys-C map Unmodified peptide Modified peptide % Modified pep. = Area (Modified pep.peak) [Area(Modified pep. Peak)+Area (Unmodified pep. Peak)] x100 7

8 Conventional Peptide Map is Used for UV Profile Confirm Identity by MS Applications for Conventional maps: Targeted quantification Sequence verification Sequence variants PTM and glycan characterization Challenges for Conventional map: Purity of peaks under UV trace Insufficient selectivity Degradation pathways 8

9 Principles of Mass Spectrometry for Mass Determination and Relative Quantitation Relative Abundance MS spectra of modified min Relative Abundance m/z Calculate the monoisotopic mass of peptides: MMMMMMMMMMMMMMMMMMMMMMMM MMMMMMMM = (mm/zz HH + ) zz Relative Abundance m/z Calculate the peak area of modified peptide Calculate the peak area of unmodified peptide PPPPPPPP AAAAAAAA MMMMMM %MMMMMM = PPPPPPPP AAAAAAAA MMMMMM + PPPPPPPP AAAAAAAA UUUUUUoooo Time (min) 9

10 Quantification of Deamidated Peptides without MS Resolution requires Chromatography GFYPSDIAVEWESNGQPENNYK Native Iso Asp-398 Asp-398 Asp-397 Iso Asp-393 Iso Asp-397 Asp /01/2013

11 All Control and Data Analysis Components are in a Single Compliant Package Chromeleon 7.2 SR2 CFR Title 21 Part 11 Compliant Package Instrument Control Attribute Quantitation New Peak Detection 11

12 Chromeleon 7.2 SR Software navigation panel Preview of integration of the selected peptide Data Analysis quantification- Pinpoint Isotopic fingerprinting /peptide I.D

13 Software: New Peak Detection Binary comparison of test sample and a reference standard starts with base peak alignment of the tryptic digest SIEVE is used to detect all of the frames (peaks) Test Sample Reference Standard New peak detected in the spiked sample Reference Standard SIEVE is able to detect new peaks 13

14 Chromeleon 7.2 SR2.0: Electronic Reporting 14

15 MAM provides selective and specific monitoring of biologically relevant attributes Conventional purity method CEX HPLC profile (mab-a) Main Peak Acidic Peaks Acidic Peaks Basic Peaks Main Peak Basic Peaks Sample % Acidic % Main % Basic mab A Traditional chromatography-based purity methods often generate individual peaks that contain multiple components 15 Multi attribute method (mab-a) Time (min) Attribute Residue Peptide Structura l Region Deamidation N53 LC 3 LC CDR1 6.2 Oxidation 2.06E9 TIC MS amg228_crt l_ % M39 H3 HC CDR1 0.2 M113 H12 HC CDR3 3.8 M253 H23 Fc 1.9 M431 H44 Fc 0.9 Mannose glycan N301 H21 Fc 10.2 Clips D276/P277 H24 Fc 0.3 MAM enables detection and quantification of specific product quality attributes.

16 Criteria for Evaluating a Peptide or Attribute using the Multi Attribute Method Development 1. Identification of the peptide/attribute is confirmed by MS2 fragmentation + orthogonal characterization methods (HILIC-MS for glycosylation) 2. The retention time window for the peptide/attribute is defined 3. Set appropriate filters and threshold for new peak in Sieve 16 Execution 1. The retention time for the peptide/attribute must be within a set retention time window (determined by characterization of the molecule) 2. The experimental mass is less than 5 ppm from the predicted mass 3. The experimental isotopic distribution fit to the theoretical must meet pre defined criteria 4. Apply filters and Threshold for new peak detection

17 Comparison of Traditional Glycan Map and MAM Excellent Agreement for Mab Fc Glycans A2G0F EU A2G1F % A1G0 A2G0 A1G0F A2G1 A2G2F M5 A1G1F A2G1 M6 M7 M Minutes 40.0% 20.0% MAM HILIC Glycan map 0.0% 17

18 MAM Provides ID Information Read out of Product Specific CDR Peptides Ensures Specificity Deamidation V H Truncation V L C H 1 Isomerization CL Cleavage Disulfide variants C H 2 Oxidation C H 3 Glycosylation Oxidation Pro Amidation C-Terminal Lys 18

19 Product Clipping: MAM vs. Reduced CE-SDS % Clipped Species Sample rce MAM* 4C, T= C, T=1 Yr C, T= 2 Yrs C, T=26 wks C, T=4 wks C, T=1 3wks C, T= 0 4C, T=1Yr 4C, T= 2Yrs 25C, T=26wks 40C, T=4wks 40C, T=13wks rce MAM* Relative levels of Clips by MAM (H9DP + H18DP clips) and reduced CE- SDS (LMW + MMW species) are in agreement 19

20 MAM has potential to Replace Several Nonattribute Specific Assays Purpose Purity - Clips Purity Charge Variants Glycans Identity Current DS Release Method rce-sds CEX-HPLC Glycan Map Immunoassay Strategy Multi-Attribute Method MS Based Peptide Map to Quantify Specific CQAs V H Deamidation V L Isomerization C L C H 2 Oxidation C H 3 C H 1 Truncation Cleavage Disulfide variants Glycosylation Oxidation Process Impurities Protein-A-ELISA Pro Amidation C-Terminal Lys Multi Attribute Method has the potential to replace several Methods and Provide more Detailed Information 20

21 QUALIFICATION/VALIDATION OF MAM 21

22 Advancing Highly Sensitive Analytical Technology When utilizing highly sensitive methods, one has to assure that you have extensive knowledge of method capability: Reproducibility/Ruggedness System Suitability Robustness Sample prep conditions Chromatography conditions MS conditions Factors that Affect Results Sensitivity, Interference, Critical method parameters Identification of Species Data Interpretation Relationship to other Methods 22

23 Qualification Design Aligned with ICH Guidelines for Purity Methods

24 Benefits of the Single Multi Attribute Method for Release: Automated quantification combining Orbitrap mass spectrometry technology and dedicated software for routine application in QC environment Science-based Provides direct measurements of attributes of criticality captured in the QTPP Scientifically superior to current methods (CE-SDS, CEX-HPLC, UV based Peptide Maps). Reduces number of release tests with the benefit of more detailed product information Flexibility Method allows for continuous input resulting from increased knowledge of the drug attributes during process development and better understanding of their criticality from clinical experience Modality independent Universal Method 24

25 Acknowledgements Jette Wypych Izydor Apostol Rohini Deshpande Sabrina Benchaar Da Ren Quanzhou Luo Wenzhou Luo Robert Hung Helen Chung Le Zhang Zhongqi Zhang Pavel Bondarenko Suminda Hapuarachchi Greg Flynn Janet Cheetham Richard Wu Tony Mire-Sluis Mike Abernathy Jason Hampson Wendy Laderach Armineh Stone Rich Rogers Nancy Nightlinger Amanda Miller Alain Balland Bob Bailey Catherine Eakin Dean Pettit Brent Kendrick Amol Prakash Scott Peterman Jenifer Sutton Christoph Nickel Ryo Komatsuzaki PacifiChem 2015, Technical Program #242 25

26 Q&A 26