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Host Cell Protein Analysis - Measuring the Forest, or Counting theTrees
- 1. Host Cell ProteinAnalysis Measuring the Forest, Or Counting the Trees? Ted Kocot IBC Life Sciences Conference 15 May 2010
- 2. 2 What is HostCell Protein? HCPs are proteins made by the host construct that are not product related.
- 3. 3 Why do wecare? Patient Safety & Product Quality HCPs may: Elicit an immune response Act as adjuvants Have enzymatic effects
- 4. 4 Why do wecare? Process Support Support development Assess performance Demonstrate comparability
- 5. 5 Why do wecare? Regulatory Expectation Release Testing Process Validation
- 6. 6 Frequently Asked Questions “Howmuch HCP is in my sample?” “Is that low enough?”
- 7. 7 The Challenges HCPs are: Proteinaceous Identicalto our products to many method Diverse E. coli have ≈4000 protein encoding genes Heterogenous A wide range of properties Rare Parts per million sensitivity is required
- 8. 8 The Challenges We solvethese problems by using an immuno- assay (ELISA) for HCPs quantitation. This takes advantage of the high Selectivity and Affinity of antibodies.
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- 12. 12 Sandwich ELISA Anti-HCP Antibodies Blocking HCPCapture Biotinylated Anti-HCP Antibodies
- 13. 13 Sandwich ELISA Anti-HCP Antibodies Blocking HCPCapture Biotinylated Anti-HCP Antibodies Streptavidin HRP Conjugate B
- 14. 14 Sandwich ELISA Anti-HCP Antibodies Blocking HCPCapture Biotinylated Anti-HCP Antibodies Streptavidin HRP Conjugate TMB Oxidization B
- 15. 15 The Assay Standard Theassay is standardized against Host Cell Protein material from a null cell culture.
- 16. 16 Antibody Production This nullcell material is also used to raise antibodies in laboratory animals.
- 17. 17 Dilutional Linearity If thereare two parts per million HCP in a 5 mg/mL solution of a product, how much in a 1 mg/mL solution?
- 18. 18 Dilutional Linearity OK, alot of discussion:
- 19. 19 An Explanation There areno antibody for these HCPs. These are antibodies against HCPs that have been removed by the process. There is insufficient antibody for these HCPs.
- 20. 20 Immunoassay Issues Only OneAntibody No Detection Non-Immunogenic HCPs No Detection Interfering Epitopes No Detection Cross Reactivity False Positives
- 21. 21 HCP ELISAs: Linearity Ina DoE exercise designed to improve HCP assay linearity it was found: Changes in the assay conditions had different effects on different sample types. Which is not too surprising, except...
- 22. 22 HCP ELISAs: Linearity Ina DoE exercise designed to improve HCP assay linearity it was found: Changes in the assay conditions had different effects on different sample types. And for one sample type: “Lower Coating Antibody concentration is better…” This is completely contrary to the conventional wisdom!
- 23. 23 HCP Distribution 25% ofall HCPs ≈20 Species 50% of all HCPs ≈80 More Species 75% of all HCPs ≈300 More Species 100% of all HCPs ≈4000 More Species
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- 29. 29 Antibody Binding Intuitively wethink of antibody-antigen binding as going to completion then stopping.
- 30. 30 Antibody Binding Intuitively wethink of antibody-antigen binding as going to completion then stopping. This is a reasonable approximation if the antibody concentration is high.
- 31. 31 Antibody Binding Intuitively wethink of antibody-antigen binding as going to completion then stopping. This is a reasonable approximation if the antibody concentration is high. But when the antibody concentration falls below the binding constant, our mental model and reality begin to diverge.
- 32. 32 The Standard Curve TheStandard Curve is the sum of the binding curves of individual antibody-HCP pairs.
- 33. 33 Individual Binding High AntibodyConc. Low Affinity High Affinity Low Antibody Conc.
- 34. 34 What is inour Sample When we consider the purification process we usually think of HCP quantitatively. 1 10 100 1000 10000 100000 Crude Final
- 35. 35 HCP Clearance -Quality But our process does not remove proteins uniformly. As we purify our product, some HCPs will be removed thoroughly. Others will co-purify. Reactor Harvest Purified Product
- 36. 36 What’s in YourSample? As the HCP profile changes, the sample response may begin to diverge from the standard response. In a bioassay it would be said that the sample is “not equivalent”.
- 37. 37 Sample Quantitation High AntibodyConc. Low Affinity High Affinity Low Antibody Conc.
- 38. 38 Going Forward So, givenall this, what practices suggest themselves? Test at a uniform product concentration (or concentrations). or Test at multiple concentrations (but always the same concentrations) and report an average.
- 39. 39 Going Forward So, givenall this, what practices suggest themselves? Run the assay as a limit test standardizing against material from an engineering run. NOTE: There are a lot of complications, both technically and logistically, inherent in this approach, so I cannot seriously recommend it. But, if dilutional linearity was our only issue, this would be the solution.
- 40. 40 Going Forward Watch outfor the things our ELISA assay might not be telling us! Western Blots – reveals a few HCPs in large abundance. Silver Stained Gels and Mass Spectrometry – reveals HCPs present in abundance but not seen by our antibody pool.
- 41. 41 Finally Watch the dose-responsecurve. If the dilutional linearity relationship suddenly changes, something is has changed, either in your assay or in the purification process.
- 42. 42 Acknowledgements Mark Abrams Sean Concannon AparnaDeora Ned Mozier Asenath Rasmussen Tom Schomogy Xing Wang Kristine Wells