Developability Assessments of Monoclonal Antibody Candidates to Minimize Aggregation During Large-Scale Ultrafiltration and Diafiltration (UF-DF) Processing.

Therapeutic proteins are subjected to a variety of stresses during manufacturing, storage or administration, that often lead to undesired protein aggregation and particle formation. Ultrafiltration-diafiltration (UF-DF) processing of monoclonal antibodies (mAbs) is one such manufacturing step that has been shown to result in such physical degradation. In this work, we explore the use of different analytical techniques and lab-scale setups as methodologies to predict and rank-order the aggregation potential of four different mAbs during large-scale UF-DF processing. In the first part of the study, a suite of biophysical techniques was applied to assess differences in their inherent bulk protein properties including conformational and colloidal stability in a PBS buffer. Additionally, the inherent interfacial properties of these mAbs in PBS were measured using a Langmuir trough technique. In the next part of the study, several different scale-down lab models were evaluated including a lab bench-scale UF-DF setup, mechanical stress (shaking/stirring) studies in vials, and application of interfacial dilatational stress using a Langmuir trough to assess protein particle formation in different UF-DF processing buffers. Taken together, our results demonstrate the ability of a Langmuir-trough methodology to accurately predict the mAb instability profile observed during large scale UF-DF processing.

Impact of Polysorbate 80 Grade on the Interfacial Properties and Interfacial Stress Induced Subvisible Particle Formation in Monoclonal Antibodies.

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80. The interfacial properties of these mAb formulations, with and without interfacial dilatational stresses, were correlated with subvisible particle count and particle size/morphology distributions as measured by Micro-flow imaging (MFI). Overall, differences in interfacial properties correlated well with protein particle formation for both molecules in the two PS80 formulations. Further, the impact of grade of PS80 on the interfacial properties and interfacial stress-induced protein particle formation depends on the adsorption kinetics of the IgG molecules as well as the concentration of the surfactant used. This study demonstrates that measuring the interfacial properties of mAb formulations can be a useful tool to predict interfacial stress induced protein particle formation in the presence of different excipients of varying quality.

Small-Scale Tools to Assess the Impact of Interfacial and Shear Stress on Biologic Drug Products.

Proper risk analysis needs to be in place to understand the susceptibility of protein to unfold and aggregate in the presence of interfacial and/or shear stress. Certain techniques, such as agitation/shaking studies, have been traditionally used to understand the impact of these stresses on the protein physical stability. However, the stresses applied in these systems are convoluted, making it difficult to define the control strategy (i.e., adjustment in process parameters to reduce foaming/bubble formation, change pump type). We have developed two small-scale tools that allow for the isolation of interfacial and shear stress, respectively. These systems, in combination with computational fluid dynamics and numerical approximations, help simulate the normal operating ranges as well as the proven acceptable ranges for different unit operations such as tangential flow filtration (TFF), mixing, and filling.

Analysis methods of polysorbate 20: A new method to assess the stability of polysorbate 20 and established methods that may overlook degraded polysorbate 20.

PURPOSE: Polysorbate 20 is a commonly used excipient in biopharmaceutical formulations, some of which may have an enzymatic activity. The action(s) of polysorbate 20 in biopharmaceutical formulations as a stabilizer require this surfactant to maintain its intact structure. This manuscript evaluates a new analytic method for the analysis of polysorbate 20 degradation in the format of a biopharmaceutical formulation and makes a comparison with several established methods of analysis. METHODS: Polysorbate 20 samples were degraded in a controlled environment utilizing the enzyme pancreatic lipase to generate degradants that included lauric acid and the sorbitan polyoxyethylene side chain. A new method was developed with sufficient sensitivity to analyze the degraded solutions. Lauric acid was derivatized with the fluorescent reagent 9-anthryldiazomethane to form 9-anthrylmethylethyl ester. The derivatized lauric acid was separated by reversed-phase chromatography and detected by fluorescence or UV spectroscopy. Three established methods utilized to measure polysorbate 20 were evaluated for their ability to detect degraded polysorbate 20. These methods were: (1) fluorescence analysis with N-phenyl-1-naphthylamine fluorescent dye; (2) UV spectroscopy with ammonium cobaltothiocyanate colorimetric reagent; and (3) nuclear magnetic resonance (NMR). RESULTS: Polysorbate 20 incubation with lipase resulted in degraded polysorbate 20 as determined by the derivatized lauric acid assay. The UV spectroscopy assay utilizing ammonium cobaltothiocyanate reagent was not able to detect the degradation of polysorbate 20 in the samples. The fluorescence method of analysis detected polysorbate 20 degradation as an approximate 50% decrease in micelles in comparison to standard nondegraded polysorbate 20 solutions. NMR analysis resulted in similar proton peak areas for both degraded and nondegraded polysorbate 20 samples. NMR spectra did contain minor differences between the samples. CONCLUSIONS: It is essential to choose the appropriate method of polysorbate 20 evaluation to assess the content, stability, and compatibility of a formulation. Current established methods to assess polysorbate 20 may overlook and do not necessarily monitor the potential degradation of this surfactant, which results in the formation of lauric acid. Because this type of degradation may occur in a formulation by an enzymatically active biopharmaceutical, a new method of analysis has been established.