The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – Part 1: Higher molecular weight species and subvisible particle formation

Abstract

Cryoconcentration upon large-scale freezing of monoclonal antibody (mAb) solutions leads to regions of different ratios of low molecular weight excipients, like buffer species or sugars, to protein. This study focused on the impact of the buffer species to mAb ratio on aggregate formation after frozen storage at −80 °C, −20 °C, and − 10 °C after 6 weeks, 6 months, and 12 months. An optimised sample preparation was established to measure T g ′ of samples with different mAb to histidine ratios via differential scanning calorimetry (DSC). After storage higher molecular weight species (HMWS) and subvisible particles (SVPs) were detected using size-exclusion chromatography (SEC) and FlowCam, respectively. For all samples, sigmoidal curves in DSC thermograms allowed to precisely determine T g ′ in formulations without glass forming sugars. Storage below T g ′ did not lead to mAb aggregation. Above T g ′, at −20 °C and − 10 °C, small changes in mAb and buffer concentration markedly impacted stability. Samples with lower mAb concentration showed increased formation of HMWS. In contrast, higher concentrated samples led to more SVPs. A shift in the mAb to histidine ratio towards mAb significantly increased overall stability. Cryoconcentration upon large-scale freezing affects mAb stability, although relative changes compared to the initial concentration are small. Storage below T g ′ completely prevents mAb aggregation and particle formation.

High concentration formulation developability approaches and considerations

“…Bluemel et al 17 report an interesting cryoconcentration effect on mAb instability. Samples with lower mAb concentration showed increased formation of high molecular weight species (i.e., aggregates).…”

Study of Oncolytic Virus Preservation and Formulation

“…They measured the water content of frozen concentrates as 20–30% [ 52 ]. At this point, the solute was highly concentrated, and the pH was drastically altered by the crystallization of the buffer components [ 53 , 54 ]. We have previously discussed the effect of pH on viral activity, see the ‘pH’ section of the article ‘Chemical Factors’ Using phosphate buffer as an example, the crystallization of buffer components in solution has been identified using low-temperature X-ray diffractometry.…”

Stochastic ice nucleation governs the freezing process of biopharmaceuticals in vials

“…Indeed, there is no consensus in literature regarding the definition of complete freezing, with some authors relying on ice mass based definitions, 16,36 and some relying on the glass transition temperature. 30,38 We interpret the distribution of the characteristic quantities among vials as measure of batch heterogeneity: vials that nucleate and solidify differently, will presumably have different mean crystal sizes and different API activity levels, 6,25,36 even if the practical relevance of these differences cannot be known a priori. We compute these distributions by running a large number of independent simulations of the process and by determining the corresponding probability density functions f nt (t), f nT (T ) and f sol (t).…”

“…[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.…”