SPIKE RECOVERY 6.


Figure 2 - Various conformational states of protein with various aggregation states of endotoxin. Question marks indicate unknown protein-endotoxin binding / interaction characteristics of various forms. Each of three (or more) protein conformation states shown may be present in various proportions for a given compound as well as various endotoxin aggregation properties. Micelles shown as cross section. MAb fi gures based upon [25].


Kerwin, B.A., Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways, Journal of Pharmaceutical Sciences 97 (2008): 2924-2935.


7. Endotoxins, Informa Healthcare, 2007, page. 169. 8.


Creation of an In-house Naturally Occurring Endotoxin Preparation for Use in Endotoxin Spiking Studies and LAL Sample Hold Time Analysis, Bowers and Tran, September/ October 2011 issue of American Pharmaceutical Review - Volume 14, Issue 6.


9. Mire-Sluis et al., Analysis and Immunogenic Potential of Aggregates and Particles, BioProcess International 9(10) November 2011


10. 11. 12.


Esfandiary et al., Characterization of Protein Aggregation & Adsorption on Prefi llable Syringe Surfaces, West Pharmaceutical Services, 2010.


Chirino and Mire-Sluis, Characterizing Biological Products and assessing comparability following manufacturing changes, Nature Biotechnology Vol. 22, No. 11, Nov. 2004.


FDA: Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997).


13. Draft guidance: Immunogenicity Assessment for Therapeutic Protein Products, Feb. 2013. 14.


Aggregates in Monoclonal Antibody Manufacturing Processes, Marı´a Va´zquez-Rey and Dietmar A. Lang, Biotechnology and Bioengineering, Vol. 108, No. 7, July, 2011.


15. Human immunoglobulin allotypes, Possible implications for immunogenicity, by Roy Jeff eris and Marie-Paule Lefranc,, July-Aug, MABS 2009; 1(4): 332-338.


16. D. Petsch, W.-D. Deckwer, and F.B. Anspach, Proteinase K Digestion of Proteins Improves Detection of Bacterial Endotoxins by the Limulus Amebocyte Lysate Assay: Application for Endotoxin Removal from Cationic Proteins, Analytical Biochemistry 259, 42-47 (1998), pg. 42-48.


17. 18.


dilemmas, it is important to remember that the best assurance of very clean drug solutions (endotoxin-free) is to ensure endotoxin removal at relevant process steps and the prevention of bioburden in manufacturing processes, the existing expectation for all cGMP drugs. Yet these same concerns apply to aid in better understanding BET testing between processing points as diff erent phases of production may have diff erent protein aggregation and thus endotoxin binding propensities.


References 1.


2.


J. Chen, “Low Endotoxin Recovery in Common Biologics Products.” Presented at the 2013 PDA Annual Meeting, Orlando, FL, April 2013.


FDA-Guidance for Industry, Pyrogen and Endotoxins Testing: Questions and Answers, Jun 2012.


3. Robert Blumenthal, BioWhittaker 1990 -Personal communication 4.


24.


Rajesh Krishnamurthy et al., Emerging Analytical Technologies for Biotherapeutics Development, BioProcess International May 2008, pg. 32-42.


5. Mire-Sluis et al., Analysis and Immunogenic Potential of Aggregates and Particles, BioProcess International 9(10) November 2011.


8. 25.


Protein Aggregation and Bioprocessing, Cromwell et al., The AAPS Journal, 2006; 8 (3) Article 66.


V. Kayser et al., Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and thiofl avin T binding, mAbs 3:4, 408-411; July/August 2011; © 2011 Landes Bioscience.


19. Demeule et al., New methods allowing the detection of protein aggregates, mAbs 1:2, 142- 150; March/April 2009; ©2009 Landes Bioscience.


20. 21. 22. 23.


Jourbert, et al., Classifi cation and Characterization of Therapeutic Antibody Aggregates, 2011, Jour. Biological Chemistry, 286, 25118-25133.


Luo et al., Chemical Modifi cations in Therapeutic Protein Aggregates Generated under Diff erent Stress Conditions, 2011, Jour. Biological Chemistry, 286, 25134-25144.


Lahlou A, et al. Mechanically-induced aggregation of the monoclonal antibody cetuximAb. Ann Pharm Fr (2009), doi:10.1016/j.pharma.2009.05.008


Aggregation Analysis of Therapeutic Proteins, Part 1, General Aspects and Techniques for Assessment Tsutomu Arakawa,\ et al., BioProcess International 4(10):42-43 (November 2006).


Chromatographic removal of endotoxin from protein solutions by polymer particles, C. Hirayama and M. Sakata, Journal of Chromatography B, 781 (2002) 419–432.


V. Kayser et al., Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and thiofl avin T binding, mAbs 3:4, 408-411; July/August 2011; © 2011 Landes Bioscience.


“...some of the most severe adverse events associated with immunogenicity occur at frequencies low enough to require very large studies.” pg. 1386 [11].


9. “The eff ect of the presence of self-associated species is not always known” [17] 10. “...the potential interaction of aggregates with surfaces, e.g., needle, tubing, column, will lead to the loss of sample and thus an inaccurate analysis” [18]. 11. “Both the excipients used during the lyophilization process and the excipients used during the reconstitution process should ensure the stability of the protein” [19].


12. “Diff erent stress treatments can generate aggregates with widely varying properties. Stressed samples can potentially have diff erent particle size distributions, particle morphology, chemical modifi cations, reversibility, percent aggregation, conformations (native-like versus unfolded), and particle surface hydrophobicity” [20].


13. “The chemical modifi cations (Met oxidation) seen with mild stirring are similar to those seen with H2 O2 9 www.americanpharmaceuticalreview.com treatment, which suggests that mild stirring stress increases interactions of the protein with air…” [21].


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