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Efficient development for multistep downstream processing of biopharmaceuticals depends on fast and robust transfer from bench to manufacturing-scale. Equivalence tests are used to assess the impact of changes in product quality and quantity during process development, optimization and production. This article presents the results of a simulation study comparing the impact of sample sizes, data distributions, variances and equivalence acceptance criteria (EAC) on the performance of equivalence tests. Our results show that a sample size of at least four in the reference group was the minimum recommended size to obtain an acceptable overall performance. Reference data should be evaluated for outliers prior to the equivalence tests and reliable test decisions are only taken with a low variance in the data of the reference group using the EAC proposed by Limentani. Considering these recommendations we obtained a reliable product yield comparability after chromatographic purification of a biopharmaceutical in bench and manufacturing-scale.

This study analyzed the efficiency of protein refolding with crossflow ultrafiltration for two distinct types of model proteins: denatured bovine α-lactalbumin and a fusion protein that comprised green fluorescent protein coupled with an engineered Npro autoprotease tag. A mechanistic model of the process dynamics that accounted for the refolding kinetics was developed and verified by comparing with experimental data. The model was used to quantify refolding performance under various operating conditions, including different denaturant removal rates, refolding durations, and protein concentrations. The performance of ultrafiltration with the fed-batch and stepwise operating modes for denaturant removal was analyzed and compared to the performance of the batch dilution method. Performance was evaluated in terms of productivity, yield, and buffer consumption. The superiority of one refolding method over another depended on the protein system. When a slow reduction in denaturant concentration suppressed protein aggregation, the best performance was achieved with the ultrafiltration system. For example, α-lactalbumin refolding with ultrafiltration achieved several-fold higher productivity and lower buffer consumption compared to refolding with the batch dilution method. A further reduction in buffer consumption was achieved with permeate recycling. Conversely, when rapid dilution of the denaturant was most efficient, a combination of batch dilution and ultrafiltration was recommended. The latter reduced the buffer consumption with permeate recycling; e.g., over 80% of the refolding buffer could be recycled during fusion protein refolding.

Inclusion bodies are a source of large quantities of protein with high purity over-expressed in Escherichia coli . Efficient solubilization is the initial step in recovering the protein. We visualized solubilization using high-resolution light and transmission electron microscopy over time and the most common solubilization agents, urea and guanidine hydrochloride, as well as sodium hydroxide. Upon solubilization, the inclusion bodies were penetrated by the solubilization agent, shrinking the densely packed cores as the protein diffused out. Despite the complex process, solubilization could be described by a homogeneous layer model with a high order of reaction. The solubilization of protein aggregates, such as inclusion bodies, does not follow the rules of ordinary solubilization processes, but is controlled by pore diffusion. Consequently, power input and mixing frequency is not relevant to the process.