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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
Acknowledgements
• Solving HCP problems is not a one-tool-fixes-all situation
• Dr. Rick Jenny and Dr. Art Rovner – Haemtech Biopharma Services – http://www.haemtechbiopharma.com/
– 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
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
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 (~106-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
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
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
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 HCP Heterogeneity
Custom CHO Cell Line
Response – Part 4 • Implement LC-MRM analysis to analyze purity on a protein-
by-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
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
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
LC-MRM – Noise and Specificity
• Interferences due to Product Protein being ~106-fold
higher concentration.
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
Response – Part 6
• Develop and validate new process-specific ELISA – Haemtech Biopharma Services
– Null cell line – same CHO cell line but no product gene
http://upload.wikimedia.org/wikipedia/commons/a/a9/ELISA.jpg
Response – Part 7
• Confirmed purity of Revised-Process Product with LC-MRM and new process-specific ELISA – Licensing process back on track
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
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
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
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