Retrospective Analysis of a Host Cell Protein Perfect Storm: Identifying Immunogenic Proteins and Fixing the Problem

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1 Retrospective Analysis of a Host Cell Protein Perfect Storm: Identifying Immunogenic Proteins and Fixing the Problem Kevin Van Cott, Associate Professor Dept. of Chemical and Biomolecular Engineering Nebraska Center for Mass Spectrometry University of Nebraska-Lincoln Lincoln, NE USA

2 Acknowledgements Solving HCP problems is not a one-tool-fixes-all situation Dr. Rick Jenny and Dr. Art Rovner Haemtech Biopharma Services 1D and 2D SDS PAGE and Western blots SEC-HPLC, IEX-HPLC, etc. Isolation of immunogenic HCPs Process-specific ELISA development and validation UNL Laura Smoyer: Research Scientist & Lab Manager

3 Background Caveats Expression in a customized, proprietary CHO cell line During process development Reliance on conventional assays: SDS-PAGE, western blots, RP-HPLC Commercial HCP ELISA Results were well within conventional HCP limits Phase III Clinical Trials <30% of subjects developed immune response Clinical Hold Regulatory Agency identify all HCPs in product, as well as immunogenic HCPs Massively parallel response between process development and analytics identify HCPs and fix the problem asap

4 Response Part 1 Retrospective analysis of Drug Product data LC-MS/MS peptide map data search against CHO proteome Both trypsin and LysC digests CHO Proteome published in 2012 Hammond et al. Biotechnol Bioeng (2012) Not useful because of Product Protein high background signal (~10 6 -fold higher concentration) HCP analysis can t be tacked onto the bottom of an assay list for a drug product; it has to be an integrated part of process development To do: HCP analysis

5 Response Part 2 Global proteomics analyses of Process Fractions Identify HCPs by LC-MS/MS analysis (trypsin digestion) Old-Process and in-progress Revised-Process samples Solution and gel-band samples (Haemtech Biopharma) Begin development of LC-MRM assay for future quantitation of individual HCPs

6 Response Part 3 Identify immunogenic HCPs Haemtech Biopharma (Essex Junction, VT, USA) Immunoglobulins from patient plasma used to isolate HCPs HCPs analyzed by LC-MS/MS to identify proteins Solution and gel-band samples LC-MRM method was updated to include immunogenic proteins Comparison with commercial CHO ELISA antibodies Commercial ELISA antibodies failed to identify 2/3 of the immunogenic HCPs identified from patient-derived antibodies Commercial HCP ELISA did not have sufficient coverage for this product

7 HCP Identification Most HCPs we identified were unique to the process/product Many HCPs were not ideal for traditional purity analysis methods Glycoproteins Spliceoforms Proteolysis Diffuse Signals in SDS-PAGE, westerns, HPLC, etc. Commercial HCP ELISA reliance Custom CHO Cell Line HCP Heterogeneity

8 Response Part 4 Implement LC-MRM analysis to analyze purity on a proteinby-protein basis Targeted and quantitative LC-MS/MS method Q1: precursor ion selection (i.e., tryptic peptide from the HCP) Q2: fragmentation Q3: production ion detection Q1/Q3 pair = transition Excellent Reviews Hoofnagle et al. (2012) Clinical Chemistry Doerr (2013) Nature Methods 2012 Method of the Year From Keck Proteomics Lab

9 LC-MRM Features & Advantages Not biased by immunization process Multiplexed method 10 s to 100 s of individual proteins can be monitored in a single injection MRM has large linear and dynamic ranges: 3 orders of magnitude Good for quantifying trace contaminants in the excess Product peptides High sensitivity Nano-LC-MS: attomoles Micro-LC-MS: femtomoles Relatively rapid method development Quantitation modes Absolute using stable isotope-labeled peptide internal standards Relative using the Product Protein

10 LC-MRM Method Development Explore all possible peptides for each protein Standard LC-MS/MS concerns with PTMs and good MS/MS features Manually confirm all peptide identities with full MS/MS spectra MRM Software: Skyline (MacCoss Lab, U. Washington) Freely available Rapid optimization of transitions Facile data processing and presentation Compatible with all major QQQ instrument platforms Narrow down to best peptides for each protein Minimum: 2 peptides/protein; 2 transitions/peptide Chromatography is important Maximize on-column resolution of product protein vs. HCP peptides Careful tracking of retention times to deal with noise and eliminate false-positives Quantitation relative to Product Protein MRM transitions for the Product Protein were monitored in each sample Easily deal with impure in-process samples as well as high-purity drug substance/product samples

11 LC-MRM Noise and Specificity Interferences due to Product Protein being ~10 6 -fold higher concentration.

12 Response Part 5 Integrate LC-MRM with Revised-Process Development Objective design a process that captures HCPs and lets the Product Protein flow through. LC-MRM-generated breakthrough curves for each HCP enabled rapid optimization of polishing step Polishing method works well for this HCP But method needed revision because of this HCP

13 Response Part 6 Develop and validate new process-specific ELISA Haemtech Biopharma Services Null cell line same CHO cell line but no product gene

14 Response Part 7 Confirmed purity of Revised-Process Product with LC- MRM and new process-specific ELISA Licensing process back on track

15 Lessons Learned from This and Other HCP Projects Integrate HCP analysis early into process development Cell line development and selection Process changes and scale-up Immunogenicity is not the only concern HCP biological function Use the power of LC-MS/MS and LC-MRM methods early Commercial HCP ELISAs trust but verify Understand limitations of immunoassays esp. immunization process Membrane proteins/fragments covered in commercial ELISAs? HCP heterogeneity via glycosylation, proteolysis, and splicing variants

16 Lessons Learned - continued Null cell line - may not be the perfect negative control, but it s the best we ve got Heterologous protein expression can alter the proteome Recombinant protein can affect chromatographic behavior via protein-protein interactions

17 LC-MS/MS & LC-MRM Recommendations HCP Identity: Confirm, confirm, confirm Don t implicitly trust Mascot or other proteomics software packages Label-free quantitation analysis is improving Optimize transitions for MRM analysis Choose peptides carefully Digest features proximity to glycosylation, adjacent Pro or acidic residues, consecutive K/R residues; proximity to N- and C-termini LC-MS features: signal specificity, retention time, potential PTMs (e.g., Met oxidation, N-term Gln, deamidation, etc.) Skyline (MacCoss Lab) ideal tool DDA data MS/MS library MRM optimization Real sample analysis

18 LC-MS/MS & LC-MRM Recommendations - continued Chromatographic separation of peptides is important Good on-column resolution = less ion suppression of HCP peptides Consistent retention times can not guarantee the HCP transitions are unique to the system; product protein peptides can provide noise Quantitation Relative to Product Protein or Absolute? Relative worked well for intermediate process samples during process revision Choose Product Protein transitions carefully Especially if MRM is used for characterization Absolute perfectly acceptable if resources are available May be required if MRM is a release assay

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