5/12/2014

Characterization of Host Cell Proteins in Biotherapeutics by Mass Spectrometry Justin Sperry, PhD Senior Principal Scientist Analytical R&D, Pfizer, Inc. BEBPA Workshop on Host Cell Protein Assays, 16-MAY-2014

Host Cell Protein Analysis

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Sensitive detection and quantitation of residual HCPs during purification process development is critical in the design of robust and well-controlled manufacturing processes that yield high quality biotherapeutics.

Topics •

HCP Characterization – Traditional vs. Emerging Technologies – LC-MS/MS Bottom-Up Approach

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Recent LC-MS/MS Applications – Virus-like Particle (E. coli): HCP  – Monoclonal Antibodies (CHO): HCP 

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Conclusions and Future Directions 2 CHO image from Wikimedia Commons

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5/12/2014

Orthogonal HCP Toolkit •

HCP ELISA is the industry gold standard – Custom reagents : HCP standard/immunogen and anti-HCP antibodies – Detects HCP as a population – Pros: semi-quantitative with good sensitivity (≤10 ng/mg) – Cons: may not detect every HCP Technique

Sensitivity (ng/mg)

ELISA*

≤10 (total HCPs)

Western Blot*

Not Quantitative

1D SDS-PAGE

~200/band

Peptide Map

~1000

CGE-Reduced

~3000

CGE-Non-reduced

~5000

New Approaches (e.g., Mass spectrometry)

TBD

*Requires specific anti-HCP immunoreagent development

1000 ng/mg = 0.1%

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Mass Spectrometry Method Attributes •

Ultrahigh-resolution mass spectrometer (Orbitrap) – Measure peptide masses at 240000 RP –