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Application and Adaptation of Platform and Alternative Purification Steps to Fc-fusion Proteins

Authored and presented by: Nathan Nicholes, Ph.D.

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Application and Adaptation of Platform and Alternative Purification Steps to Fc-fusion Proteins

  1. 1. Confidential ACS 2019 BIOT Section Application and adaptation of platform and alternative purification steps to Fc-fusion proteins Nathan Nicholes Ph.D. Scientist II, DSPD
  2. 2. Confidential 2 KBI Biopharma: Who We Are • Analytical & Formulation Services, Microbial, Mammalian & Cell Therapy CDMO • CDMO services first offered in 2004 • Acquired by JSR Corp in 2015 as part of JSR Life Sciences • Highly experienced Executive & Management Teams • Excellent Track Record & Client Satisfaction
  3. 3. Confidential 3 Durham, NC (2004) Mammalian • Clinical & Commercial cGMP Manufacturing • Analytical, Formulation, Stability & QC • Mass Spec Core Facility • Cell Based Assays Boulder, CO (2014) Microbial • Strain Development • Process & Analytical Development • cGMP Manufacturing and QC Services • Analytical, Formulation, Stability • Particle Characterization Core • Modeling and Simulation RTP & Venture Center, NC (2013) Mammalian • Cell Line Development • Process & Analytical Development • Process Characterization • Small scale Process ValidationThe Woodlands, TX (2017) Cell Therapy • Process and Analytical Development • cGMP Manufacturing & Testing • Cell Based Assays San Diego, CA (2017) Alliance Protein Labs • Analytical Technologies • Leading AUC expertise KBI Sites KBI North American Locations March 2018 Louisville, CO (2018) Elion Labs • Analytical Technologies • Leading Biophysical Chara
  4. 4. Confidential 4 SelexisKBI Selexis, Geneva, Switzerland (2017- an affiliate of KBI) Best in class cell line development & variant screening technologies KBI Leuven, BE (2018) Analytical, Formulation and QC services for GMP & non-GMP product testing KBI European Locations
  5. 5. Inspected by EMA/MHRA and the FDA Commercial drug substance and drug product release testing for >10 years COMMERCIAL READY AND INSPECTED QUALITY SYSTEMS HIGHLY SUCCESSFUL cGMP MANUFACTURING First CDMO to implement 2,000L single-use cGMP operations Single-use clinical and commercial manufacturing trains (three 2,000L SUBs) Focus on the science for mAb and non-mAb platform development Well versed first-in-human and late-stage commercialization development Innovation in new equipment and technology adoption WORLD CLASS PROCESS AND ANALYTICAL DEVELOPMENT Industry leading array of analytical equipment and scientific expertise 14 years of leadership in formulation development More than 200 active product stability studies (GMP & non- GMP) WORLD LEADERS IN ANALYTICAL SERVICES Extensive Success in Analytical Services, Process Development and Manufacturing
  6. 6. Confidential 6 Fc-Fusion Protein Diversity Platform Development Strategies Platform Adaptations to address purification challenges -resHCP clearance -Aggregation during low pH VI -UF/DF low concentration pH offsets Conclusions Technical Presentation Outline
  7. 7. Confidential 7 Fc-Fusion proteins are a significantly more diverse class of molecules than mAbs or bispecific antibodies Hydrophobicity, pI, Charge Distribution, Glycosylation etc. Application of Platform-Based Expedited DSPD approach to enable rapid IND filing entails some risks due to greater molecule diversity Process development anticipated to utilize platform conditions for ProA, low pH VI, VF and UFDF. Development and optimization anticipated for polishing steps This presentation will highlight differences between Fc-Fusion proteins that fit platform purification steps well vs. those that did not and alternative purification strategies for more challenging molecules. Fc-Fusion Proteins and Platform DSPD Development
  8. 8. Confidential 8 Platform relies on ProA for capture from harvest Low pH VI, Virus Filtration and UF/DF are expected to require minimal optimization Platform polishing steps rely on pI of molecule being significantly different from most resHCP (pI > 7.0) Optimization and robustness evaluation expected for polishing chromatography steps mAb/Fc-Fusion Platform Purification Evaluation Viral Inactivation AEX Based Polishing Step CEX Based Polishing Step Virus Filtration ProA Capture Ultrafiltration/Diafiltration
  9. 9. Confidential 9 This presentation will discuss specific challenges that we commonly encounter in adapting platform purification steps to more unusual Fc-fusion proteins -resHCP removal over capture and VI -Detergent Viral Inactivation -AEX Gradient vs. Isocratic Elution Development -UF/DF Low concentration pH offsets mAb/Fc-Fusion Platform Purification Development Challenges Viral Inactivation AEX Based Polishing Step CEX Based Polishing Step Virus Filtration ProA Capture Ultrafiltration/Diafiltration
  10. 10. Confidential 10 resHCP levels post capture can vary widely from molecule to molecule As a CDMO, we see a variety of different cell lines that vary in both productivity and purity Typical range for ProA eluate is 1000 – 20,000 ppm resHCP ProA eluate resHCP levels setup clearance requirements for subsequent steps resHCP clearance achieved in platform purification through AEX or AEX/MMC polishing step Post load modulator washes on ProA Redundant clearance on CEX or second polishing step Platform process resHCP clearance
  11. 11. Confidential 11 Lower resHCP levels can be cleared with more common arginine-based intermediate washes More aggressive ProA washes can remove more resHCP however they also lower yields and can impact product quality Yield losses occur as product is washed from the column during the modulator wash phase resHCP and Yields for two Fc-fusions with different ProA modulators
  12. 12. Confidential 12 Platform purification process employs low pH viral inactivation post capture Low pH viral inactivation requires that the product be stable at pH conditions that are low enough to inactivate enveloped viruses Product instability at low pH is a more frequent problem with Fc-fusion proteins and requires implementation of alternative viral inactivation strategy Detergent Viral Inactivation can be applied pre or post ProA capture Application before ProA capture typically requires larger volumes of detergent but also reduces detergent levels in subsequent process steps Viral Inactivation Development
  13. 13. Confidential 13 resHCP levels were significantly lower with detergent viral inactivation of the harvest material resHCP clearance is likely due to detergent disrupting product/HCP interactions on the column Step yields were similar despite the presence of detergent in harvest material Impact of Detergent Viral Inactivation on resHCP levels and yields post ProA
  14. 14. Confidential 14 AEX is a critical part of the platform purification process for Viral Clearance as well as removal of resDNA and resHCP Platform mAb/Fc-fusion AEX purification typically operates in flow through mode and can achieve high product loading on the resin (50 - 300 g/L) It is not always possible to operate at a pH that will allow for AEX operation in flowthrough for acidic Fc-fusion proteins and achieve a robust separation between product and resHCP Column loading capacities are significantly lower for AEX in bind and elute mode AEX resin selection and chromatography development
  15. 15. Confidential 15 Isocratic elution is preferred due to lower operational risk and simpler process characterization for late phase process development For early phase clinical supply, however, gradient elution can provide both higher yields and better resHCP clearance relative to isocratic elution conditions. AEX Elution Mode (Isocratic vs. Gradient)
  16. 16. Confidential 16 Platform UFDF conditions assume that buffer exchange occurs without selective partitioning of ions across the membrane Gibbs-Donnan equilibrium effects are typically observed at high protein concentrations for mAbs (>100 g/L) We have observed that some Fc-fusion proteins show pH offsets between the diafiltration buffer and the final formulated BDS at significantly lower concentrations (≤50 g/L) UFDF Development Challenges with Fc-Fusion Proteins
  17. 17. Confidential 17 “…when nondiffusible charged molecules, such as proteins, are participating in an equilibrium process such as UF/DF, the nonideality of the process, due to the unequal partitioning of charged solutes across the membrane, yields an unequal distribution of electrolytes and therefore results in significant differences in molar buffer and excipient concentrations between the final formulation and the DF buffer. This phenomenon has been attributed to the Donnan and volume exclusion effect.” Abel J et al. J Pharm Sci 107:1296-1303, 2018 UFDF Development Challenges with Fc-Fusion Proteins
  18. 18. Confidential 18 “The Donnan effect can cause a large offset in pH from the target value established with the diafiltration buffer during the concentration and diafiltration of charged proteins with ultrafiltration membranes. For neutral formulations, the pH will typically increase above the diafiltration buffer pH for basic monoclonal antibodies and decline below the diafiltration buffer pH for acidic Fc-fusion proteins.” From Bolton GR et al. Biotechnol. Prog., 27(1):140-152, 2011 UFDF Development Challenges with Fc-Fusion Proteins
  19. 19. Confidential 19 Certain formulation buffer/molecule pairings result in pH offsets between the diafiltration buffer and the final formulated material Options for addressing these offsets include Evaluate product stability at pH values around the pH of the final formulated BDS Increasing buffer ion strength Assessing pH offset with diafiltration buffer Redeveloping formulation to a formulation system that does not show an offset (not always an option) UFDF pH offsets at low protein concentrations
  20. 20. Confidential 20 Diversity of Fc-fusion proteins can complicate the application of platform purification strategies for rapid process development Detergent inactivation in harvest provided additional, unanticipated reductions in resHCP in the ProA eluate pool Gradient elution on AEX can provide higher yields and better product quality relative to isocratic elution conditions although it does entail some operational risk Some Fc-fusion proteins can show unusual behavior during UF/DF at lower concentrations that requires modification of the TFF operation or formulation buffer Conclusions
  21. 21. Confidential Questions? Nate Nicholes You can also connect with me on LinkedIn Visit us at