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The optimization and control of cell free protein synthesis (CFPS) presents an ongoing challenge due to the complex synergies and nonlinearities that cannot be fully explained in first principle models. This article explores the use of multivariate statistical tools for analyzing data sets collected from the CFPS of Cereulide monoclonal antibodies. During the collection of these data sets, several of the process parameters were modified to investigate their effect on the end‐point product (yield). Through the application of principal component analysis and partial least squares (PLS), important correlations in the process could be identified. For example, yield had a positive correlation with pH and NH3 and a negative correlation with CO2 and dissolved oxygen. It was also found that PLS was able to provide a long‐term prediction of product yield. The presented work illustrates that multivariate statistical techniques provide important insights that can help support the operation and control of CFPS processes.

The impact of two different quality feeds, derived using two different harvest clarification processes, on protein A periodic counter-current chromatography (PCC) design and performance is investigated. Data from batch experiments were input into a model to design optimal PCC operating parameters specific to each feed material. The two clarification methods were: depth filtration using a wetlaid matrix which has Q-functionality; and a combination of depth filtration and chromatographic clarification, using a Q-functional nonwoven with a high anion exchange capacity (Emphaze™ AEX Hybrid Purifier) in which key impurities such as host cell DNA (HCDNA) and host cell proteins (HCP) are removed. The model predicted 34% better productivity for the chromatographically clarified cell culture fluid (CCCF) using a 4 column system, and productivity gains of 28% using only 3 columns enabling the option to simplify the protein A PCC strategy. Experimental validation of the predicted optimized PCC operating parameters using industrially relevant monoclonal antibody (mAb) CCCF feedstock over 100 cycles showed productivity gains of 49% for the chromatographically clarified material. HCP concentration was 11-fold lower, and HCDNA concentration was reduced by 4.4 Log Reduction Value (LRV) in the protein A PCC eluates. This work, therefore, demonstrates that the removal of HCDNA and HCP during clarification is an effective strategy for improving protein A PCC performance. This was achieved using the Emphaze™ AEX Hybrid Purifier which can be easily incorporated into a batch or continuous process, in a scalable fashion, without adding additional separate unit operations. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1380-1392, 2018.

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018.

Determination of copy number for integrated expression units in selected P. pastoris clones. Description of the methodology used for quantification of copy number for expression cassettes integrated in selected P. pastoris clones. The method is based on real time PCR amplification of zeocin gene. (DOCX 186 kb)

Serum IgG reactivity against the â emptyâ K1-K1 HBc VLPs and against irrelevant antigens (Cytomegalovirus antigen, purified EBV capsid protein, Toxoplasma gondii antigen). Reactivity of mouse sera with carrier HBc-derived VLPs (positive control) in comparison with irrelevant antigens (negative control) demonstrating specificity of ELISA method used. (PPTX 91 kb)