The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – part 2: Aggregate formation and oxidation

Abstract

We examined the impact of monoclonal antibody (mAb) and buffer concentration, mimicking the cryoconcentration found upon freezing in a 2 L bottle, on mAb stability during frozen storage. Upon cryoconcentration, larger protein molecules and small excipient molecules freeze-concentrate differently, resulting in different protein to stabiliser ratios within a container. Understanding the impact of these shifted ratios on protein stability is essential. For two mAbs a set of samples with constant mAb (5 mg/mL) or buffer concentration (medium histidine/adipic acid) was prepared and stored for 6 months at −10 °C. Stability was evaluated via size-exclusion chromatography, flow imaging microscopy, UV/Vis spectroscopy at 350 nm, and protein A chromatography. Dynamic light scattering was used to determine k D values. Soluble aggregate levels were unaffected by mAb concentration, but increased with histidine concentration. No trend in optical density could be identified. In contrast, increasing mAb or buffer concentration facilitated the formation of subvisible particles. A trend towards attractive protein-protein interactions was seen with higher ionic strength. MAb oxidation levels were negatively affected by increasing histidine concentration, but became less with higher mAb concentration. Small changes in mAb and buffer composition had a significant impact on stability during six-month frozen storage. Thus, preventing cryoconcentration effects in larger freezing containers may improve long-term stability.

High concentration formulation developability approaches and considerations

“…The authors went on to show that the protein and small excipients freeze-concentrate differently, resulting in different local protein to stabilizer ratios within a container, ultimately leading to mAb instability under these storage conditions. 18 Interestingly, Bluemel et al 19 have recently reported on the use of computational fluid dynamic (CFD) simulations of mAb solutions. They conclude that CFD simulations are a promising tool to describe large-scale freezing and thawing of mAb solutions and could potentially save time and resources.…”

Stochastic ice nucleation governs the freezing process of biopharmaceuticals in vials

“…[16][17][18][19] Solidification is initiated by ice nucleation, which is a stochastic process, 16,17,[20][21][22][23][24] leading to variability in freezing behavior among vials even if their thermal environment is identical. 16,18,19,23,25,26 Based on these considerations, freezing may be characterized by three quantities, which are conceptually linked to most of the known degradation mechanisms for frozen biopharmaceuticals: [7][8][9][10]18,19,[27][28][29][30][31] These are the nucleation time, i.e. the duration of the liquid state; the nucleation temperature, which is interpreted as predictor for the ice crystal morphology in the frozen product; 19,27 and the solidification time, which is the duration of the partially frozen state and connected to the phenomenon of freeze-concentration.…”

“…the duration of the liquid state; the nucleation temperature, which is interpreted as predictor for the ice crystal morphology in the frozen product; 19,27 and the solidification time, which is the duration of the partially frozen state and connected to the phenomenon of freeze-concentration. [28][29][30][31] In case a drug product exhibits a relevant degradation mechanism related to freezing, these three quantities should be considered during development, implying that all vials meet qualified target ranges with sufficient probability.…”