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Analytical Platform for Monitoring Aggregation of Monoclonal Antibody Therapeutics

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Abstract

Purpose

To develop an analytical platform for the estimation as well as characterization of aggregates over the complete size spectrum (from invisible monomer to visible precipitates).

Methods

Two mAb samples were incubated at 30°C in different buffer systems of protein A chromatography for observing degradation due to aggregation. The aggregation in these samples was quantified by size exclusion chromatography (SEC), dynamic light scattering (DLS), and micro flow imaging (MFI).

Results

The results obtained from various characterization tools were analysed in various size ranges - size exclusion chromatography (SEC) (1 nm - 25 nm), dynamic light scattering (DLS) (10 nm - 5 μm), and micro flow imaging (MFI) (2 μm - 300 μm). Since each characterization tool covers a particular size range, data from multiple tools was collected in the “handover” regions to demonstrate accuracy of the platform.

Conclusions

Based on the observations from the experiments, an analytical platform has been proposed covering the whole size spectrum that would be of utility to those engaged in formulation development as well as other aspects related to stability of biotherapeutic products.

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Abbreviations

AF4:

Asymmetric Flow Field Flow Fractionation

AUC:

Analytical Ultra Centrifugation

CC:

Coulter Counter

CD:

Circular Dichroism

CQA:

Critical Quality Attributes

DLS:

Dynamic Light Scattering

EMA:

European Medicines Agency

FDA:

Food and Drug Administration

FTIR:

Fourier Transform Infra-Red Spectroscopy

HMW:

High Molecular Weight

LO:

Light Obscuration

mAb:

Monoclonal Antibody

MALS:

Multi Angle Light Scattering

MFI:

Micro Flow Imaging

NMR:

Nuclear Magnetic Resonance

NTA:

Nanoparticle Tracking Analysis

SDS-PAGE:

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

SEC:

Size Exclusion Chromatography

SEM-EDX:

Scanning Electron Microscopy – Energy Dispersive X-ray Spectroscopy

SLS:

Static Light Scattering

TEM-EDX:

Transmission Electron Microscopy – Energy Dispersive X-ray Spectroscopy

USP:

United States Pharmacopeia

UV-Visible Spectroscopy:

Ultra Violet –Visible Spectroscopy

References

  1. Nicolaides NC, Sass PM, Grasso L. Monoclonal antibodies: a morphing landscape for therapeutics. Drug Dev Res. 2006;67(10):781–9.

    Article  CAS  Google Scholar 

  2. Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC. Monoclonal antibody successes in the clinic. Nat Biotechnol. 2005;23(9):1073–8.

    Article  CAS  PubMed  Google Scholar 

  3. ICH Harmonised Tripartite Guideline, Specifications: Test procedures and acceptance criteria for biotechnological/biological products Q6B, 1999. Available from: http://www.ich.org/LOB/media/MEDIA4986.pdf https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q6B/Step4/Q6B_Guideline.pdf. Accessed 1 Sept 1999

  4. Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J. 2006;8(3):E501–7.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm. 2005;289(1–2):1–30.

    Article  CAS  PubMed  Google Scholar 

  6. Wang W, Roberts CJ, editors. Aggregation of therapeutic proteins. New Jersey: Wiley; 2010.

    Google Scholar 

  7. Joshi V, Shivach T, Kumar V, Yadav N, Rathore AS. Avoiding antibody aggregation during processing: establishing hold times. Biotechnol J. 2014;9(9):1195–205.

    Article  CAS  PubMed  Google Scholar 

  8. Shukla AA, Hubbard B, Tressel T, Guhan S, Low D. Downstream processing of monoclonal antibodies—application of platform approaches. J Chromatogr B. 2007;848(1):28–39.

    Article  CAS  Google Scholar 

  9. Ravuluri S, Bansal R, Chhabra N, Rathore AS. Kinetics and characterization of non-enzymatic fragmentation of monoclonal antibody therapeutics. Pharm Res. 2018;35(7):142.

    Article  PubMed  CAS  Google Scholar 

  10. Vázquez-Rey M, Lang DA. Aggregates in monoclonal antibody manufacturing processes. Biotechnol Bioeng. 2011;108(7):1494–508.

    Article  PubMed  CAS  Google Scholar 

  11. CMC Biotech Working Group. A-Mab: A case study in bioprocess development. Emeryville: CASSS; 2009.

    Google Scholar 

  12. Carpenter JF, Randolph TW, Jiskoot W, Crommelin DJ, Middaugh CR, Winter G, et al. Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality. J Pharm Sci. 2009;98(4):1201–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Huang CT, Sharma D, Oma P, Krishnamurthy R. Quantitation of protein particles in parenteral solutions using micro-flow imaging. J Pharm Sci. 2009;98(9):3058–71.

    Article  CAS  PubMed  Google Scholar 

  14. Ishikawa T, Ito T, Endo R, Nakagawa K, Sawa E, Wakamatsu K. Influence of pH on heat-induced aggregation and degradation of therapeutic monoclonal antibodies. Biol Pharm Bull. 2010;33(8):1413–7.

    Article  CAS  PubMed  Google Scholar 

  15. Rathore AS, Joshi V, Yadav N. Aggregation of monoclonal antibody products: formation and removal. Biopharm International. 2013;26(3):40–5.

    CAS  Google Scholar 

  16. Carpenter JF, Randolph TW, Jiskoot W, Crommelin DJ, Middaugh CR, Winter G. Potential inaccurate quantitation and sizing of protein aggregates by size exclusion chromatography: essential need to use orthogonal methods to assure the quality of therapeutic protein products. J Pharm Sci. 2010;99(5):2200–8.

    Article  CAS  PubMed  Google Scholar 

  17. Fekete S, Beck A, Veuthey JL, Guillarme D. Theory and practice of size exclusion chromatography for the analysis of protein aggregates. J Pharm Biomed Anal. 2014;101:161–73.

    Article  CAS  PubMed  Google Scholar 

  18. Cromwell ME, Hilario E, Jacobson F. Protein aggregation and bioprocessing. AAPS J. 2006;8(3):E572–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cao S, Pollastrini J, Jiang Y. Separation and characterization of protein aggregates and particles by field flow fractionation. Curr Pharm Biotechnol. 2009;10(4):382–90.

    Article  CAS  PubMed  Google Scholar 

  20. Liu J, Andya JD, Shire SJ. A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregation. AAPS J. 2006;8(3):E580–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Singh SK, Afonina N, Awwad M, Bechtold-Peters K, Blue JT, Chou D, et al. An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics. J Pharm Sci. 2010;99(8):3302–21.

    Article  CAS  PubMed  Google Scholar 

  22. Narhi LO, Schmit J, Bechtold-Peters K, Sharma D. Classification of protein aggregates. J Pharm Sci. 2012;101(2):493–8.

    Article  CAS  PubMed  Google Scholar 

  23. Mahler HC, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci. 2009;98(9):2909–34.

    Article  CAS  PubMed  Google Scholar 

  24. Bickel F, Herold EM, Signes A, Romeijn S, Jiskoot W, Kiefer H. Reversible NaCl-induced aggregation of a monoclonal antibody at low pH: characterization of aggregates and factors affecting aggregation. Eur J Pharm Biopharm. 2016;107:310–20.

    Article  CAS  PubMed  Google Scholar 

  25. Mahler HC, Müller R, Friess W, Delille A, Matheus S. Induction and analysis of aggregates in a liquid IgG1-antibody formulation. Eur J Pharm Biopharm. 2005;59(3):407–17.

    Article  CAS  PubMed  Google Scholar 

  26. Hernandez R. Continuous manufacturing: a changing processing paradigm. Biopharm International. 2015; 28(4).

  27. Joshi V, Shivach T, Yadav N, Rathore AS. Circular dichroism spectroscopy as a tool for monitoring aggregation in monoclonal antibody therapeutics. Anal Chem. 2014;86(23):11606–13.

    Article  CAS  PubMed  Google Scholar 

  28. Den Engelsman J, Garidel P, Smulders R, Koll H, Smith B, Bassarab S, et al. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Pharm Res. 2011;28(4):920–33.

    Article  CAS  Google Scholar 

  29. He F, Phan DH, Hogan S, Bailey R, Becker GW, Narhi LO, et al. Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence. J Pharm Sci. 2010;99(6):2598–608.

    Article  CAS  PubMed  Google Scholar 

  30. Kong J, Yu S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin. 2007;39(8):549–59.

    Article  CAS  PubMed  Google Scholar 

  31. Wen ZQ. Raman spectroscopy of protein pharmaceuticals. J Pharm Sci. 2007;96(11):2861–78.

    Article  CAS  PubMed  Google Scholar 

  32. Philo JS. A critical review of methods for size characterization of non-particulate protein aggregates. Curr Pharm Biotechnol. 2009;10(4):359–72.

    Article  CAS  PubMed  Google Scholar 

  33. Bansal R, Dhawan S, Chattopadhyay S, Maurya GP, Haridas V, Rathore AS. Peptide Dendrons as thermal-stability amplifiers for immunoglobulin G1 monoclonal antibody biotherapeutics. Bioconjug Chem. 2017;28(10):2549–59.

    Article  CAS  PubMed  Google Scholar 

  34. Guttman A, Rathore AS, Krull IS. Bioanalytical tools for the characterization of biologics and biosimilars. LC GC North America. 2012;30(5):1–5.

    Google Scholar 

  35. Mendhe R, Rathore AS, Krull IS. Analytical tools for enabling process analytical technology applications in biotechnology. LC GC North America. 2012;30(1).

  36. Singla A, Bansal R, Joshi V, Rathore AS. Aggregation kinetics for IgG1-based monoclonal antibody therapeutics. AAPS J. 2016;18(3):689–702.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Morris AM, Watzky MA, Finke RG. Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature. Biochim Biophys Acta Protein Proteomics. 2009;1794(3):375–97.

    Article  CAS  Google Scholar 

  38. Roberts CJ. Non-native protein aggregation kinetics. Biotechnol Bioeng. 2007;98(5):927–38.

    Article  CAS  PubMed  Google Scholar 

  39. Watzky MA, Morris AM, Ross ED, Finke RG. Fitting yeast and mammalian prion aggregation kinetic data with the Finke− Watzky two-step model of nucleation and autocatalytic growth. Biochemistry. 2008;47(40):10790–800.

    Article  CAS  PubMed  Google Scholar 

  40. Li Y, Lubchenko V, Vekilov PG. The use of dynamic light scattering and Brownian microscopy to characterize protein aggregation. Rev Sci Instrum. 2011;82(5):053106.

    Article  PubMed  CAS  Google Scholar 

  41. Ahrer K, Buchacher A, Iberer G, Josic D, Jungbauer A. Analysis of aggregates of human immunoglobulin G using size-exclusion chromatography, static and dynamic light scattering. J Chromatogr A. 2003;1009(1–2):89–96.

    Article  CAS  PubMed  Google Scholar 

  42. USP <788> Particulate Matter in Injections. USP 35; U.S. Pharmacopeial Convention: Rockville, MD, 2012; 339–342. Available from: https://www.uspnf.com/sites/default/files/usp_pdf/EN/USPNF/revisionGeneralChapter788.pdf. Accessed 21 May 2001

  43. Sharma DK, King D, Oma P, Merchant C. Micro-flow imaging: flow microscopy applied to sub-visible particulate analysis in protein formulations. AAPS J. 2010;12(3):455–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Rubinstein M, Colby RH. Polymer physics, vol. 23. New York: Oxford university press; 2003.

    Google Scholar 

  45. Van der Kant R, Karow-Zwick AR, Van Durme J, Blech M, Gallardo R, Seeliger D, et al. Prediction and reduction of the aggregation of monoclonal antibodies. J Mol Biol. 2017;429(8):1244–61.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Hamrang Z, Rattray NJ, Pluen A. Proteins behaving badly: emerging technologies in profiling biopharmaceutical aggregation. Trends Biotechnol. 2013;31(8):448–58.

    Article  CAS  PubMed  Google Scholar 

  47. Kameoka D, Masuzaki E, Ueda T, Imoto T. Effect of buffer species on the unfolding and the aggregation of humanized IgG. J Biochem. 2007;142(3):383–91.

    Article  CAS  PubMed  Google Scholar 

  48. Nobbmann U, Connah M, Fish B, Varley P, Gee C, Mulot S, et al. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol Genet Eng Rev. 2007;24(1):117–28.

    Article  CAS  PubMed  Google Scholar 

  49. Hawe A, Hulse WL, Jiskoot W, Forbes RT. Taylor dispersion analysis compared to dynamic light scattering for the size analysis of therapeutic peptides and proteins and their aggregates. Pharm Res. 2011;28(9):2302–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ye H. Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography–multiangle laser light scattering. Anal Biochem. 2006;356(1):76–85.

    Article  CAS  PubMed  Google Scholar 

  51. Gabrielson JP, Brader ML, Pekar AH, Mathis KB, Winter G, Carpenter JF, et al. Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity. J Pharm Sci. 2007;96(2):268–79.

    Article  CAS  PubMed  Google Scholar 

  52. Hong P, Koza S, Bouvier ES. A review size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J Liq Chromatogr Relat Technol. 2012;35(20):2923–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The authors would like to thank Protein Simple, San Jose, CA, USA, for providing us the access to the MFI for particle size analysis. This work was funded by the Centre of Excellence for Biopharmaceutical Technology grant from Department of Biotechnology, Government of India (number BT/COE/34/SP15097/2015). The authors declare no financial or commercial conflict of interest.

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  1. Department of Chemical Engineering, DBT Center of Excellence for Biopharmaceutical Technology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, 110016, India

    Rohit Bansal, Surbhi Gupta & Anurag S. Rathore

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  1. Rohit Bansal
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Correspondence to Anurag S. Rathore.

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Bansal, R., Gupta, S. & Rathore, A.S. Analytical Platform for Monitoring Aggregation of Monoclonal Antibody Therapeutics. Pharm Res 36, 152 (2019). https://doi.org/10.1007/s11095-019-2690-8

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