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

Thank you for your attention

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