(W1230-01-02) Impact of Amino Acids as Excipients in Biologic Drug Products Formulation Development

Purpose: Amino acids are widely used excipients as protein stabilizers impacting critical quality attributes including protein aggregation, particle formation, and viscosity mainly through a variety of molecular interactions. Subsequently, amino acid concentration is critical for protein’s stability affecting the quality, safety, efficacy, and shelf-life of protein-based drug products. In this work, as case studies, we explored the effects of different ‘Generally Recognized as Safe (GRAS)’ listed amino acid categories by Food and Drug Administration (FDA) on two human monoclonal antibodies– mAb1 (hIgG1) and mAb2 (hIgG4) in solution using different characterization techniques (1). Our work highlights that, at the studied conditions, non-polar amino acid formulations demonstrated improved thermal, chemical and colloidal stability of the mAbs with the least propensity for aggregation. Our case studies aim to provide insight on rational selection of different amino acids during biotherapeutic drug product development.

Methods: Formulation screening study for mAbs was performed using different categories of amino acids at a range of concentrations in an optimal buffer condition. The mAbs in optimal buffer condition without amino acid was established as a negative control. All filtered formulations were evaluated at initial timepoint (T0) along with stressed conditions of five freeze-thaw cycles and 1–month storage at 40°C. The physiochemical properties and stability of mAbs were assessed using visual inspection, protein concentration by slope spectroscopy, turbidity measurement, solubility by polyethylene glycol precipitation (PEG) assay, particulate formation by microflow imaging, colloidal stability and molecular interaction by dynamic light scattering, thermal stability by differential scanning calorimetry (DSC), and purity by size exclusion - high performance liquid chromatography (SE-HPLC).

Results: Visual inspection of mAb1 samples in non-polar amino acids formulations demonstrated clear solutions whereas highly opalescent solutions were observed for charged amino acids formulations indicating physical instability of mAb1 that could be attributed to attractive molecular interactions in solution or aggregates formation (2). All mAb2 formulations were clear, non-opalescent solutions. Consequently, the protein concentrations were lower than target for the opalescent mAb1 formulations which further decreased upon stressed conditions. For mAb1, turbidity and hydrodynamic radius (Rh) of non-polar amino acid formulations decreased compared to the negative controls, but significantly increased for charged amino acids formulations indicating aggregation propensity and increased protein-protein interactions in the solution. PEG precipitation assay for mAb1 indicated lowest solubility for formulations with charged amino acids compared to the magnitude depicting higher solubility for non-polar amino acid formulations. The sub-visible particle concentration per mL for mAb1 formulations with charged amino acid was significantly higher compared to non-polar amino acid formulations indicating effect of amino acids on colloidal stability of these two mAbs. For mAb1 formulations containing non-polar amino acids, high purity by SE-HPLC was acquired at initial timepoint (T0). On contrary, the mAb1 formulations with charged amino acids demonstrated lower purity at T0 with significant aggregation upon stress; thereby demonstrating high aggregation propensity. DSC showed that with increasing concentrations of non-polar amino acids, thermal stability increased for both mAb1 and mAb2 molecules. In contrast, detrimental effect on thermal stability of both mAbs was observed in charged amino acids formulations. Diffusion interaction coefficient (kD) indicates protein-protein interactions with negative kD values suggesting strong molecular attraction and positive kD values indicating repulsion between molecules (3,4). In both cases of mAb1 and mAb2, the non-polar amino acid formulations showed repulsive molecular interactions whereas the charged amino acid formulations showed strong attractive molecular interactions in the solution.

Conclusion: Using biophysical techniques, we successfully characterized two case studies of mAbs by employing common amino acids as excipients thereby identifying the importance of non-polar amino acids, challenges posed by charged amino acids, and the effect of their concentrations in developing biologics formulations. The investigations presented here aims to provide insight in selection of suitable amino acids to make informed decision to advance biologics formulation development.

References: 1. US Food and Drug Administration; Code of Federal Regulations (CFR); Chapter I; US Code Title 21; Volume 3 [21CFR172.320], Silver Springs (MD).
2. Raut A.S, Kalonia D.S. Opalescence in monoclonal antibody solutions and its correlation with intermolecular interactions in dilute and concentrated solutions. J Pharm Sci.104(4):1263-74 (2015).
3. Xu A.Y, Clark N.J, Pollastrini J, Espinoza M, Kim H.J, Kanapuram S, Kerwin B, Treuheit M.J, Krueger S, McAuley A, Curtis J.E. Effects of monovalent salt on protein-protein interactions of dilute and concentrated monoclonal antibody formulations. Antibodies. 11(24) (2022).
4. Tomar D.S, Kumar S, Singh S.K, Goswami S, Li L. Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development. mAbs. 8(2):216-28 (2016).

Acknowledgements: Meghan Mcgarry for providing technical support. Project supported by Chemistry Manufacturing and Controls (CMC), Teva Pharmaceuticals, USA.