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Surfaces Affect Screening Reliability in Formulation Development of Biologics

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Abstract

Purpose

The ability to predict an antibody’s propensity for aggregation is particularly important during product development to ensure the quality and safety of therapeutic antibodies. We demonstrate the role of container surfaces on the aggregation process of three mAbs under elevated temperature and long-term storage conditions in the absence of mechanical stress.

Methods

A systematic study of aggregation is performed for different proteins, vial material, storage temperature, and presence of surfactant. We use size exclusion chromatography and micro-flow imaging to determine the bulk concentration of aggregates, which we combine with optical and atomic force microscopy of vial surfaces to determine the effect of solid-liquid interfaces on the bulk aggregate concentration under different conditions.

Results

We show that protein particles under elevated temperature conditions adhere to the vial surfaces, causing a substantial underestimation of aggregation propensity as determined by common methods used in development of biologics. Under actual long-term storage conditions at 5°C, aggregate particles do not adhere to the surface, causing an increase in bulk concentration of particles, which cannot be predicted from elevated temperature screening tests by common methods alone. We also identify specific protein – surface interactions which promote oligomer formation in the nanometre range.

Conclusions

Special care should be taken when interpreting size exclusion and particle count data from stability studies if different temperatures and vial types are involved. We propose a novel combination of methods to characterise vial surfaces and bulk solution for a full understanding of protein aggregation processes in a sample.

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Abbreviations

AFM:

Atomic force microscopy

ECD:

Equivalent circular diameter

GuHCl:

Guanidine hydrochloride

mAb:

Monoclonal antibody

MFI:

Micro-flow imaging

MWCO:

Molecular weight cutoff

PETG:

Polyethylene terephthalate glycol

PS80:

Polysorbate 80

SEC:

Size exclusion chromatography

UPW:

Ultrapure water

References

  1. Vazquez-Rey M, Lang DA. Aggregates in monoclonal antibody manufacturing processes. Biotechnol Bioeng. 2011;108:1494–508.

    Article  CAS  Google Scholar 

  2. Kueltzo LA, Wang W, Randolph TW, Carpenter JF. Effects of Solution Conditions, Processing Parameters, and Container Materials on Aggregation of a Monoclonal Antibody during Freeze-Thawing. J Pharm Sci. 2008;97:1801–12.

    Article  CAS  Google Scholar 

  3. Sathish JG, Sethu S, Bielsky MC, de Haan L, French NS, Govindappa K, et al. Challenges and approaches for the development of safer immunomodulatory biologics. Nat Rev Drug Discov. 2013;12(4):306–24.

    Article  CAS  Google Scholar 

  4. Chi EY, Krishnan S, Randolph TW, Carpenter JF. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res. 2003;20(9):1325–34.

    Article  CAS  Google Scholar 

  5. Cohen SIA, Vendruscolo M, Dobson CM, Knowles TPJ. From macroscopic measurements to microscopic mechanisms of protein aggregation. J Mol Biol. 2012;421:160–71.

    Article  CAS  Google Scholar 

  6. Ratanji KD, Derrick JP, Dearman RJ, Kimber I. Immunogenicity of therapeutic proteins: Influence of aggregation. J Immunotoxicol. 2014;11(2):99–109.

    Article  CAS  Google Scholar 

  7. Fathallah AM, Chiang M, Mishra A, Sandeep K, Xue L, Middaugh CR, et al. The Effect of Small Oligomeric Protein Aggregates on the Immunogenicity of Intravenous and Subcutaneous Administered Antibodies. Pharm Biotechnol. 2015;104:3691–702.

    CAS  Google Scholar 

  8. Wang X, Das KD, S. K S, Kumar S. Potential aggregation prone regions in biotherapeutics. mAbs. 2009;1(3):254–67.

    Article  Google Scholar 

  9. Arosio P, Rima S, Morbidelli M. Aggregation Mechanism of an IgG2 and two IgG1 Monoclonal Antibodies at low pH: From Oligomers to Larger Aggregates. Pharm Res. 2013;30:641–54.

    Article  CAS  Google Scholar 

  10. Brummitt RK, Nesta DP, Chang L, Chase SF, Laue TM, Roberts CJ. Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions: Part 1. Unfolding, Colloidal Interactions, and Formation of High-Molecular-Weight Aggregates. J Pharm Sci. 2011;100:2087–103.

    Article  CAS  Google Scholar 

  11. Yageta S, Lauer TM, Trout BL, Honda S. Conformational and Colloidal Stabilities of Isolated Constant Domains of Human Immunoglobulin G and Their Impact on Antibody Aggregation under Acidic Conditions. Mol Pharm. 2015;12:1443–55.

    Article  CAS  Google Scholar 

  12. Barnett GV, Drenski M, Razinkov V, Reed WF, Roberts CJ. Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering. Anal Biochem. 2016;511:80–91.

    Article  CAS  Google Scholar 

  13. Kim N, Remmele RL Jr, Liu D, Razinkov VI, Fernandez EJ, Roberts CJ. Aggregation of anti-streptavidin immunoglobulin gamma-1 involves Fab unfolding and competing growth pathways mediated by pH and salt concentration. Biophys Chem. 2013;172:26–36.

    Article  CAS  Google Scholar 

  14. Imamura H, Honda S. Kinetics of Antibody Aggregation at Neutral pH and Ambient Temperatures Triggered by Temporal Exposure to Acid. J Phys Chem B. 2016;120:9581–9.

    Article  CAS  Google Scholar 

  15. Nicoud L, Arosio P, Sozo M, Yates A, Norrant E, Morbidelli M. Kinetic Analysis of the Multistep Aggregation Mechanism of Monoclonal Antibodies. J Phys Chem B. 2014;118:10595–10,606.

    Article  CAS  Google Scholar 

  16. Smoluchowski M. Drei Vorträge über Diffusion, Brownsche Molekularbewegung und Koagulation von Kolloidteilchen. Zeitschrift für Physik. 1916;17:557–71, 585–599.

    Google Scholar 

  17. Wattis JA. An introduction to mathematical models of coagulation–fragmentation processes: A discrete deterministic mean-field approach. Phys D. 2006;222:1–20.

    Article  Google Scholar 

  18. Kryven I, Lazzari S, Storti G. Population Balance Modeling of Aggregation and Coalescence in Colloidal Systems. Macromol Theory Simul. 2014;23:170–81.

    Article  CAS  Google Scholar 

  19. Koepf E, Schroeder R, Brezesinski G, Friess W. The film tells the story: Physical-chemical characteristics of IgG at the liguid-air interface. Euro J Pharm Biopharm. 2017;119:396–407.

    Article  CAS  Google Scholar 

  20. Ghazvini S, Kalonia C, Volkin DB, Dhar P. Evaluating the Role of the Air-Solution Interface on the Mechanism of Subvisible Particle Formation Caused by Mechanical Agitation for an IgG1 mAb. J Pharm Sci. 2016;105:1643–56.

    Article  CAS  Google Scholar 

  21. Shieh IC, Patel AR. Predicting the Agitation-Induced Aggregation of Monoclonal Antibodies Using Surface Tensiometry. Mol Pharm. 2015;12:3184–93.

    Article  CAS  Google Scholar 

  22. Mehta SB, Lewus R, Bee JS, Randolph TW, Carpenter JF. Gelation of a Monoclonal Antibody at the Silicone Oil-Water Interface and Subsequent Rupture of the Interfacial Gel Results in Aggregation and Particle Formation. Pharm Biotechnol. 2015;104:1282–90.

    CAS  Google Scholar 

  23. Sediq AS, Duijvenvoorde RBV, Jiskoot W, Nejadnik MR. No Touching! Abrasion of adsorbed protein is the root cause of subvisible particle formation during stirring. J Pharm Sci. 2016;105:519–29.

    Article  CAS  Google Scholar 

  24. P. T, N. H, N. DP and R. CJ, “Protein Adsorption, Desorption, and Aggregation Mediated by Solid-Liquid Interfaces,” Pharm Biotechnol. 2015;104, (6), 1946–1959.

  25. X. H, Z. X, G. C and L. JR, “Orientation of a Monoclonal Antibody Adsorbed at the Solid/Solution Interface: A Combined Study Using Atomic Force Microscopy and Neutron Reflectivity,” Langmuir. 2006;22, (14), 6313–6320.

  26. F. CW and W. ME, “Antibody adsorption and orientation on hydrophobic surfaces,” Langmuir. 2012; 28, (3), 1765–1774.

  27. Randolph TW, Schiltz E, Sederstrom D, Steinmann D, Mozziconacci O, Schoneich C, et al. Do not drop: mechanical schock in vials causes cavitation, protein aggregation, and particle formation. Pharm Biotechnol. 2015;104:602–11.

    CAS  Google Scholar 

  28. Torisu T, Maruno T, Hamaji Y, Ohkubo T. Synergistic Effect of Cavitation and Agitation on Protein Aggregation. J Pharm Sci. 2017;106:521–9.

    Article  CAS  Google Scholar 

  29. Wang S, Wu G, Zhang X, Tian Z, Zhang N, Hu T, et al. Stabilizing two IgG1 monoclonal antibodies by surfactants: Balance between aggregation prevention and structure perturbation. Euro J Pharm Biopharm. 2017;114:263–77.

    Article  CAS  Google Scholar 

  30. Kalonia C, Kumru OS, Prajapati I, Mathaes R, Engert J, Zhou S, et al. Calculating the Mass of Subvisible Protein Particles with Improved Accuracy Using Microflow Imaging Data. Pharm Biotechnol. 2015;104(2):536–47.

    CAS  Google Scholar 

  31. M. Zidar, D. Kuzman and R. M, “Characterisation of protein aggregation with the Smoluchowski coagulation approach for use in biopharmaceuticals,” Soft Matter. 2018; 14, 6001–6012.

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ACKNOWLEDGMENTS AND DISCLOSURES

M.R. acknowledges funding from Lek Pharmaceuticals d.d. under contract BIO17/2016 and from Slovenian Research Agency ARRS grants L1-8135, P1-0099, and P1-0340. M.Z. and D.K. acknowledge funding from program BioPharm.Si project co-funded by Republic of Slovenia - Ministry of Education, Science and Sport - and European union - European regional development fund.

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Authors and Affiliations

  1. Novartis Global Drug Development/Technical Research & Development, Technical Development Biosimilars, Lek Pharmaceuticals d. d., Kolodvorska 27, Mengeš, Slovenia

    Mitja Zidar & Drago Kuzman

  2. Jožef Stefan Institute, Jamova 39, 1000, Ljubljana, Slovenia

    Gregor Posnjak, Igor Muševič & Miha Ravnik

  3. Faculty of Mathematics and Physics, University of Ljubljana, Jadranska 19, 1000, Ljubljana, Slovenia

    Igor Muševič & Miha Ravnik

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  1. Mitja Zidar
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  2. Gregor Posnjak
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  3. Igor Muševič
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  5. Drago Kuzman
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Correspondence to Drago Kuzman.

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Zidar, M., Posnjak, G., Muševič, I. et al. Surfaces Affect Screening Reliability in Formulation Development of Biologics. Pharm Res 37, 27 (2020). https://doi.org/10.1007/s11095-019-2733-1

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