Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system

Abstract

ObjectivesThe effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic.ResultsBuffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme.ConclusionsPeptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s10529-015-2014-y) contains supplementary material, which is available to authorized users.

The effect of formulation variables on protein stability and integrity of a model IgG4 monoclonal antibody and translation to formulation of a model ScFv

“…The effect of formulation variables (pH, buffer composition, glycine and NaCl concentration, time and temperature of accelerated stability studies) on antibody solubility/aggregation and activity were investigated. In order to investigate the effect of these variables on protein aggregation and stability whilst limiting the potential number of conditions to investigate, a Plackett-Burman Experimental Design was implemented (Supplementary Tables 1 and 2) in a manner to that previously described (Gourbatsi et al 2016).…”

“…Heavy and light chain analysis of the model mAb by mass spectrometry. Mass spectrometry analysis was undertaken as previously described (Gourbatsi et al 2016). Table S2) resulted in the majority of mAb precipitating out to form a gel like aggregate (data not shown).…”

“…Further, molecules that are constituted of domains or sections of antibody have now also been developed and require different active protein drug concentrations, excipients and additives. Formulation development of recombinant proteins is usually undertaken using a screening approach whereby excipients and additives are changed in order to find a combination that provides the desired stability profile (Almeida et al 2017;Gourbatsi et al 2016). Here, we report on the investigation of the effect of different formulations variables on protein integrity as measured by aggregation, activity, structural and chemical stability using a model mAb IgG4 molecule.…”

Aggregation Time Machine: A Platform for the Prediction and Optimization of Long-Term Antibody Stability Using Short-Term Kinetic Analysis

“…mAb stability studies are therefore usually shortened by performing experiments under stress conditions (40 °C) that accelerate the aggregation process. Typically, different formulations are tested in parallel for the differences in aggregation propensity, and the final formulation is developed iteratively based on several stability studies . However, it is not clear how accurately do such studies reflect the aggregation process at the intended low temperature storage conditions. ,, Ultimately, the final formulation is confirmed by analyzing the samples stored at 5 °C, which takes as long as the declared shelf life of the therapeutic antibody.…”