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Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches

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Abstract

Purpose

Characterizing submicron protein particles (approximately 0.1–1μm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS).

Methods

Resistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment.

Results

A method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4 × 107-1 × 1011 particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering.

Conclusion

RPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.

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Abbreviations

AF4:

Asymmetrical flow field fractionation

AUC:

Analytical ultracentrifugation

DLS:

Dynamic light scattering

LO:

Light obscuration

mAb:

Monoclonal antibody

MFI:

Micro flow imaging

NTA:

Nanoparticle tracking analysis

PBS:

Phosphate buffer saline

PS-80:

Polysorbate-80

RMM:

Resonant mass measurement

RPS:

Resistive pulse sensing

SAXS:

Small angle x-ray scattering

SEC:

Size exclusion chromatography

SEC-MALS:

Size exclusion chromatography with multi-angle light scattering

References

  1. Das TK. Protein particulate detection issues in biotherapeutics development--current status. AAPS PharmSciTech. 2012;13(2):732–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  3. Cheung CS, Anderson KW, Patel PM, Cade KL, Phinney KW, Turko IV. A new approach to quantification of mAb aggregates using peptide affinity probes. Sci Rep. 2017;7:42497.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Arvinte T, Palais C, Green-Trexler E, Gregory S, Mach H, Narasimhan C, et al. Aggregation of biopharmaceuticals in human plasma and human serum: implications for drug research and development. MAbs. 2013;5(3):491–500.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Boll B, Bessa J, Folzer E, Rios Quiroz A, Schmidt R, Bulau P, Finkler C, Mahler HC, Huwyler J, Iglesias A, Koulov AV. Extensive chemical modifications in the primary protein structure of IgG1 subvisible particles are necessary for breaking immune tolerance. Mol Pharm. 2017;14(4):1292–9.

    Article  CAS  PubMed  Google Scholar 

  6. Joubert MK, Hokom M, Eakin C, Zhou L, Deshpande M, Baker MP, et al. Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses. J Biol Chem. 2012;287(30):25266–79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rombach-Riegraf V, Karle AC, Wolf B, Sorde L, Koepke S, Gottlieb S, et al. Aggregation of human recombinant monoclonal antibodies influences the capacity of dendritic cells to stimulate adaptive T-cell responses in vitro. PLoS One. 2014;9(1):e86322.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Ahmadi M, Bryson CJ, Cloake EA, Welch K, Filipe V, Romeijn S, Hawe A, Jiskoot W, Baker MP, Fogg MH. Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics. Pharm Res. 2015;32:1383–94.

    Article  CAS  PubMed  Google Scholar 

  9. Pharmacopeia US. USP: <788> Particular Matter in Injections. Revision Bulletin. 2011.

  10. Ripple DC, Hu Z. Correcting the Relative Bias of Light Obscuration and Flow Imaging Particle Counters. Pharm Res. 2016;33(3):653–72.

    Article  CAS  PubMed  Google Scholar 

  11. Joubert MK, Luo Q, Nashed-Samuel Y, Wypych J, Narhi LO. Classification and characterization of therapeutic antibody aggregates. J Biol Chem. 2011;286(28):25118–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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 

  13. 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 

  14. Bria CR, Jones J, Charlesworth A, Ratanathanawongs Williams SK. Probing Submicron Aggregation Kinetics of an IgG Protein by Asymmetrical Flow Field-Flow Fractionation. J Pharm Sci. 2016;105(1):31–9.

    Article  CAS  PubMed  Google Scholar 

  15. Hawe A, Romeijn S, Filipe V, Jiskoot W. Asymmetrical flow field-flow fractionation method for the analysis of submicron protein aggregates. J Pharm Sci. 2012;101(11):4129–39.

    Article  CAS  PubMed  Google Scholar 

  16. Wagner M, Holzschuh S, Traeger A, Fahr A, Schubert US. Asymmetric flow field-flow fractionation in the field of nanomedicine. Anal Chem. 2014;86(11):5201–10.

    Article  CAS  PubMed  Google Scholar 

  17. 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 

  18. Weinbuch D, Cheung JK, Ketelaars J, Filipe V, Hawe A, den Engelsman J, et al. Nanoparticulate Impurities in Pharmaceutical-Grade Sugars and their Interference with Light Scattering-Based Analysis of Protein Formulations. Pharm Res. 2015;32:2419–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tian X, Nejadnik MR, Baunsgaard D, Henriksen A, Rischel C, Jiskoot W. A Comprehensive Evaluation of Nanoparticle Tracking Analysis (NanoSight) for Characterization of Proteinaceous Submicron Particles. J Pharm Sci. 2016;105(11):3366–75.

    Article  CAS  PubMed  Google Scholar 

  20. Filipe V, Hawe A, Jiskoot W. Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res. 2010;27(5):796–810.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhou C, Krueger AB, Barnard JG, Qi W, Carpenter JF. Characterization of Nanoparticle Tracking Analysis for Quantification and Sizing of Submicron Particles of Therapeutic Proteins. J Pharm Sci. 2015;104(8):2441–50.

    Article  CAS  PubMed  Google Scholar 

  22. Bai K, Barnett GV, Kar SR, Das TK. Interference from proteins and surfactants on particle size distributions measured by nanoparticle tracking analysis (NTA). Pharm Res. 2017;34:800–8.

    Article  CAS  PubMed  Google Scholar 

  23. Rios Quiroz A, Finkler C, Huwyler J, Mahler HC, Schmidt R, Koulov AV. Factors Governing the Accuracy of Subvisible Particle Counting Methods. J Pharm Sci. 2016;105(7):2042–52.

    Article  CAS  PubMed  Google Scholar 

  24. Rios Quiroz A, Lamerz J, Da Cunha T, Boillon A, Adler M, Finkler C, et al. Factors Governing the Precision of Subvisible Particle Measurement Methods - A Case Study with a Low-Concentration Therapeutic Protein Product in a Prefilled Syringe. Pharm Res. 2016;33(2):450–61.

    Article  CAS  PubMed  Google Scholar 

  25. Yang L, Yamamoto T. Quantification of Virus Particles Using Nanopore-Based Resistive-Pulse Sensing Techniques. Front Microbiol. 2016;7:1500.

    PubMed  PubMed Central  Google Scholar 

  26. Maas SL, Broekman ML, de Vrij J. Tunable Resistive Pulse Sensing for the Characterization of Extracellular Vesicles. Methods Mol Biol. 2017;1545:21–33.

    Article  PubMed  Google Scholar 

  27. Fraikin JL, Teesalu T, McKenney CM, Ruoslahti E, Cleland AN. A high-throughput label-free nanoparticle analyser. Nat Nanotechnol. 2011;6(5):308–13.

    Article  CAS  PubMed  Google Scholar 

  28. Berne, BJ, Pecora, R. Dynamic light scattering: with applications to chemistry, biology, and physics. Mineola: Dover Publications; 2000.

    Google Scholar 

  29. Amin S, Barnett GV, Pathak JA, Roberts CJ, Sarangapani PS. Protein aggregation, particle formation, characterization & rheology. Curr Opin Colloid Interface Sci. 2014;19(5):438–49.

    Article  CAS  Google Scholar 

  30. Gupta A, Eral HB, Hatton TA, Doyle PS. Controlling and predicting droplet size of nanoemulsions: scaling relations with experimental validation. Soft Matter. 2016;12(5):1452–8.

    Article  CAS  PubMed  Google Scholar 

  31. Li Y, Roberts CJ. Lumry-Eyring nucleated-polymerization model of protein aggregation kinetics. 2. Competing growth via condensation and chain polymerization. J Phys Chem B. 2009;113(19):7020–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ejima D, Tsumoto K, Fukada H, Yumioka R, Nagase K, Arakawa T, et al. Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies. Proteins. 2007;66(4):954–62.

    Article  CAS  PubMed  Google Scholar 

  33. Hari SB, Lau H, Razinkov VI, Chen S, Latypov RF. Acid-induced aggregation of human monoclonal IgG1 and IgG2: molecular mechanism and the effect of solution composition. Biochemistry. 2010;49(43):9328–38.

    Article  CAS  PubMed  Google Scholar 

  34. Hawe A, Kasper JC, Friess W, Jiskoot W. Structural properties of monoclonal antibody aggregates induced by freeze-thawing and thermal stress. Eur J Pharm Sci. 2009;38(2):79–87.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Biologics Characterization and Analytical Development, Bristol-Myers Squibb, Hopewell, New Jersey, USA

    Gregory V. Barnett, Julia M. Perhacs, Tapan K. Das & Sambit R. Kar

Authors
  1. Gregory V. Barnett
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  2. Julia M. Perhacs
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  3. Tapan K. Das
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  4. Sambit R. Kar
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Correspondence to Sambit R. Kar.

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Barnett, G.V., Perhacs, J.M., Das, T.K. et al. Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches. Pharm Res 35, 58 (2018). https://doi.org/10.1007/s11095-017-2306-0

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  • DOI: https://doi.org/10.1007/s11095-017-2306-0

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