Computational fluid dynamic simulations of temperature, cryoconcentration, and stress time during large-scale freezing and thawing of monoclonal antibody solutions.

PURPOSE: Large-scale freezing and thawing experiments of monoclonal antibody (mAb) solutions are time and material consuming. Computational Fluid Dynamic (CFD) modeling of temperature, solute composition as well as the stress time, defined as the time between start of freezing and reaching Tg' at any point in the container, could be a promising approach to ease and speed up process development. METHODS: Temperature profiles at six positions were recorded during freezing and thawing of a 2L rectangular bottle and compared to CFD simulations via OpenFOAM. Furthermore, cryoconcentration upon freezing and concentration gradients upon thawing of a mAb solution were predicted and the stress time calculated. RESULTS: Temperature profiles during freezing were accurately matched by the CFD simulation. Thawing time was only 45 min to 60 min longer in the model. The macroscopic cryoconcentration of the mAb was also matched by the simulation; only a highly concentrated region in the top and a diluted core in the geometrical centre of the 2 L bottle were not well reflected in the simulation. The concentration gradient after thawing obtained by simulation as well agreed with the experimental result. In addition, CFD simulations allowed to extract the global temperature distribution, the formation of ice, and thus the distribution of stress in the freezing liquid. CONCLUSION: CFD simulations via OpenFOAM are a promising tool to describe large-scale freezing and thawing of mAb solutions and can help to generate a deeper understanding and to improve testing of the robustness of the processes.

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

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 Tg' 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 Tg' in formulations without glass forming sugars. Storage below Tg' did not lead to mAb aggregation. Above Tg', 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 Tg' completely prevents mAb aggregation and particle formation.

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

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 kD 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.

Evaluation of Two Novel Scale-Down Devices for Testing Monoclonal Antibody Aggregation During Large-Scale Freezing.

There is a need for representative small volume devices that reflect monoclonal antibody (mAb) aggregation during freezing and thawing (FT) in large containers. We characterised two novel devices that aim to mimic the stress in rectangular 2 L bottles. The first scale-down device (SDD) consists of a 125 mL bottle surrounded by a 3D printed cover that manipulates heat exchange. The second device, a micro scale-down device (mSDD), adapts cooling and heating of 10 mL vials to extend stress time. MAb aggregation upon repeated FT was evaluated considering formation of higher molecular weight species, subvisible particles, and the increase in hydrodynamic radius, polydispersity index, and optical density at 350 nm. Three different mAb solutions were processed. Both an unshielded 125 mL bottle and the SDD can be used to predict aggregation during FT in 2 L bottles. In specific cases the unshielded 125 mL bottle underestimates whereas the SDD slightly overestimates soluble aggregate formation. The mSDD increases aggregation compared to 10 mL vials but is less representative than the SDD. Ultimately, both SDDs enable characterisation of protein sensitivity to large-scale FT with two orders of magnitude less volume and are superior to simply using smaller bottles.

Scaling Down Large-Scale Thawing of Monoclonal Antibody Solutions: 3D Temperature Profiles, Changes in Concentration, and Density Gradients.

PURPOSE: Scale-down devices (SDD) are designed to simulate large-scale thawing of protein drug substance, but require only a fraction of the material. To evaluate the performance of a new SDD that aims to predict thawing in large-scale 2 L bottles, we characterised 3D temperature profiles and changes in concentration and density in comparison to 125 mL and 2 L bottles. Differences in diffusion between a monoclonal antibody (mAb) and histidine buffer after thawing were examined. METHODS: Temperature profiles at six distinct positions were recorded with type T thermocouples. Size-exclusion chromatography allowed quantification of mAb and histidine. Polysorbate 80 was quantified using a fluorescent dye assay. In addition, the solution's density at different locations in bottles and the SDD was identified. RESULTS: The temperature profiles in the SDD and the large-scale 2 L bottle during thawing were similar. Significant concentration gradients were detected in the 2 L bottle leading to marked density gradients. The SDD slightly overestimated the dilution in the top region and the maximum concentrations at the bottom. Fast diffusion resulted in rapid equilibration of histidine. CONCLUSION: The innovative SDD allows a realistic characterisation and helps to understand thawing processes of mAb solutions in large-scale 2 L bottles. Only a fraction of material is needed to gain insights into the thawing behaviour that is associated with several possible detrimental limitations.

Cryoconcentration and 3D Temperature Profiles During Freezing of mAb Solutions in Large-Scale PET Bottles and a Novel Scale-Down Device.

PURPOSE: Small-scale models that simulate large-scale freezing of bulk drug substance of biopharmaceuticals are highly needed to define freezing and formulation parameters based on process understanding. We evaluated a novel scale-down device (SDD), which is based on a specially designed insulation cover, with respect to changes in concentration after freezing, referred to as cryoconcentration, and 3D temperature profiles. Furthermore, the effect of the initial monoclonal antibody (mAb) concentration on cryoconcentration was addressed. METHODS: 2 L and 125 mL bottles were utilized. Temperatures were mapped using type T thermocouples. Frozen blocks were cut and mAb and histidine concentrations were analysed by HPLC. In addition, concentration- and temperature-dependent viscosities were measured. RESULTS: 3D freezing profiles in the SDD were comparable to large-scale bottles. The SDD accurately predicted cryoconcentration of both mAb and histidine of large-scale freezing. Concentric changes in concentration were evident as well as an unforeseen diluted core at the last point to freeze. At low initial mAb concentration cryoconcentration was substantial, while high initial mAb concentration suppressed cryoconcentration almost completely. CONCLUSION: The novel SDD gives detailed insights into large-scale freezing of mAb solutions using only a fraction of the simulated volume. It is a promising material- and cost-saving tool to understand large-scale freezing processes.