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Quantitative Imaging for Particle Analysis of Biopharmaceuticals Michelle Kelleher, Ph.D

Quantitative Imaging for Particle Analysis of Biopharmaceuticals Michelle Kelleher, Ph.D May 8 th , 2014. Micro-flow Imaging For Biopharmaceuticals. The challenge of protein aggregation The regulatory environment MFI technology solves challenges of detecting protein aggregates

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Quantitative Imaging for Particle Analysis of Biopharmaceuticals Michelle Kelleher, Ph.D

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  1. Quantitative Imaging for Particle Analysis of Biopharmaceuticals Michelle Kelleher, Ph.D May 8th, 2014

  2. Micro-flow Imaging For Biopharmaceuticals • The challenge of protein aggregation • The regulatory environment • MFI technology solves challenges of detecting protein aggregates • MFI easily identifies key quality and safety attributes

  3. Proteins Can Aggregate to Form Particles Proteins are fragile Change conformation Native protein Unfolded protein Start aggregate Amorphous aggregate Clump and form particles Difficult to measure translucent particles Protein Particulate Protein particle formation Adapted from TK Das , AAPS Pharm Sci Tech (2012)

  4. Sub-visible Particles Impact Safety and Efficacy • Even trace levels of SVPs can impact subsequent stability of protein solutions • SVPs serve as adjuvants and promote immunogenicity • Loss of therapeutic efficacy John Carpenter presentation at Japan Particle Seminar 2013. Sub-visible Particles: Impacts on Aggregation Pathways, Adverse Immunogenicity and Regulatory Expectations for Therapeutic Proteins.

  5. Development and Manufacturing Processes Can Induce Aggregation • Heat • Freeze-Thaw • Exposure to Light • Protein Concentration • Formulation Change • Extractables/Leachables • Chemical Modification • Mechanical Stress • Surface Effects & Interfaces • Nano-particle Nucleation Sites How to Predict Impact • Fermentation & Harvesting • Formulation • Drug Product Manufacture • Storage • Shipping • Final Preparation ? *Adapted from: Linda O. Narhi, Shawn Cao, Yijia Jiang. Current State of Sub-Visible Particle Analysis in Protein Products. USP Workshop on Particle Size, December 2010.

  6. Aggregation Can Occur Throughout Production “Protein particle formation is ubiquitous in the production, shipping, storage and delivery of therapeutic proteins” John Carpenter, University of Colorado John Carpenter presentation at Japan Particle Seminar 2013. Sub-visible Particles: Impacts on Aggregation Pathways, Adverse Immunogenicity and Regulatory Expectations for Therapeutic Proteins.

  7. Early SVPs Impact Product Quality “In some formulations, the presence of early SVPs leads to protein aggregates in manufacturing.” . *Mire-Sluis, A., Cherney, B., Madsen, R., Polozova, A., Rosenberg, A., Smith, H., Arora, T., Narhi, L. Analysis and Immunogenic Potential of Aggregates and Particles. A practical approach Part 1.BioProc Intern. 2011, 9(10):38-47

  8. Sub-Visible Particles May Predict Amount of Visible Particles Linda O. Narhi, Shawn Cao, Yijia Jiang. Current State of Sub-Visible Particle Analysis in Protein Products. USP Workshop on Particle Size, December 2010.

  9. “Need to measure particles throughout development” Maria Toler, Pfizer Maria Toler Thesis Dissertation: Techniques To Improve Characterization of Protein Particles in Biologic Drug Products, 2012.

  10. Regulatory Agencies Request SVP Data • Data on subvisible particles from 2 – 10 µm using a quantitative method: • “Assessment should be made of the range and levels of subvisible particles (2-10 microns) present in therapeutic protein products initially and over the course of the shelf life.” Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products. February 2013.

  11. Upcoming Regulatory Guidelines for SVPs • USP <787> Subvisible particulate matter in parenteral biopharmaceuticals - official announcement expected August 2014 • USP <1787> Measurement of subvisible particulate matter in therapeutic protein injections – expected release in late 2014 • Will define specifications for 2-5 and 5-10 microns

  12. Gap In Pharmacopeial Methods:Detection of Translucent Particles Light Obscuration Fails to Detect Particle Increase • Light Obscuration • Micro-Flow Imaging t0 t20 t20 t0 New Analytical Methods for the Assessment of the Physical Stability of Therapeutical Proteins Veronika M Spalthoff, Thesis Dissertation, 2013

  13. A Better Option - Micro-Flow Imaging

  14. MFI Addresses Gap In Pharmacopeial Methods

  15. MFI Is A Powerful Imaging Tool • Direct, quantitative imagingof particle size, count, shape • Detects subvisible protein aggregates 2-10 µm in size • Shape parameters evaluate differences in particle type

  16. MFI Particle Analysis Enables a Safe and High Quality Biopharmaceutical • Sub-population changes • Process monitoring • Improved efficacy • Better products

  17. Particle Characterization Case Studies – Product Stability and Safety • Excipient impact on particle formation • Choice of pump affects particle formation • Differences in protein aggregation for β-interferon analogues correlates with immunogenicity Development Manufacturing Quality Control

  18. Formulation Development Detecting Formulation Instability

  19. MFI Detects Aggregation Changes Better Than Other Techniques • “Because MFI was able to detect changes in aggregate levels, the team was able to look at the effect of excipients as stabilizers that prevent aggregate formation, such as polysorbate 80.” • Shannon Southall, GSK Southall, S., Ketkar, A., Brisbane, C., Nesta, D. Particle Analysis as a Formulation Development Tool. Am. Pharma. Rev. 2011, Sept/Oct.: 61-66.

  20. Excipient Impact on SVP Formation from Aggregation PS-80 Most Effective at Inhibiting Particle Formation Southall, S., Ketkar, A., Brisbane, C., Nesta, D. Particle Analysis as a Formulation Development Tool. Am. Pharma. Rev. 2011, Sept/Oct.: 61-66.

  21. Manufacturing Development Impact of Manufacturing Pumps

  22. Pump Shear Stress Increases Particle Formation • Different pumps vary in shear stress (Ceramic vs. Steel) • Particles increase with greater pump shear stress (90 mg/ml IgG1) Refs: Wuchner and Volkin et al. IBC Conference- Formulation Strategies for Protein Therapeutics (2010) &Koulov et al. BPI Europe (2010)

  23. Safety Assessment SVP Correlation to Immunogenicity

  24. β-interferon Therapeutics Vary In Particle Shape Avonex Extavia* Silicone & few aggregates Lowest immunogenicity Elongated fibrous particles of IFN-β Betaseron Rebif Large dense clusters of IFN-β Many protein aggregates adsorbed onto silicone oil *Extavia is the same product as Betaseron, marketed under a different name. Adapted from Figure 5 in Ref: J Barnard, K Babcock and J Carpenter, J of Pharmaceutical Sciences, 2013; 102(3):915–28.

  25. Protein Aggregation Correlates With Immunogenicity Adapted from Table 3 & Figure 5 in Ref: J Barnard, K Babcock and J Carpenter, J of Pharmaceutical Sciences, 2013; 102(3):915–28.

  26. Image is Everything

  27. Useful References • JAMES G. BARNARD, KEN BABCOCK, JOHN F. CARPENTER, Characterization and Quantitation of Aggregates and Particles in Interferon-β Products: Potential Links Between Product Quality Attributes and Immunogenicity, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013 • AMY S. ROSENBERG, DANIELA VERTHELYI, BARRY W. CHERNEY, Managing Uncertainty: A Perspective on Risk Pertaining to Product Quality Attributes as They Bear on Immunogenicity of Therapeutic Proteins, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 10, OCTOBER 2012 • SATISH K. SINGH, NATALIYA AFONINA, MICHEL AWWAD, KAROLINE BECHTOLD-PETERS, JEFFREY T. BLUE, DANNY CHOU, MARY CROMWELL, HANS-JUERGEN KRAUSE, HANNS-CHRISTIAN MAHLER, BRIAN K. MEYER, LINDA NARHI, DOUG P. NESTA, THOMAS SPITZNAGEL, An Industry Perspective on the Monitoring of Subvisible Particles as a Quality Attribute for Protein Therapeutics, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 8, AUGUST 2010 • SARAH ZOLLS, RUEDEEPORN TANTIPOLPHAN, MICHAEL WIGGENHORN, GERHARD WINTER, WIM JISKOOT, WOLFGANG FRIESS, ANDREA HAWE, Particles in Therapeutic Protein Formulations, Part 1: Overview of Analytical Methods, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012 • INTERVIEW WITH 2014 PRESENTER DR. SATISH SINGH, PFIZER RESEARCH FELLOW, GROUP LEADER, AND MEMBER OF THE 787 EXPERT PANEL FOR THE U.S. PHARMACOPEIAL CONVENTION (USP) • http://www.pegsummit.com/PEGS_Content.aspx?id=136339 • International consortium for Innovation and quality in pharmaceutical development. IQ consortium considers subvisible particles as a topic of interest. • http://iqconsortium.org/

  28. Q&A Michelle Kelleher Field Applications Scientist Michelle.Kelleher@proteinsimple.com

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