Use of quality by design principles for development of upstream process control strategy

A systematic approach was developed using the QbD (Quality by Design) principles to study and characterize monoclonal antibody upstream production processes. This approach comprises of risk assessment of upstream process parameters, small scale model development and qualification, process characterization as well as the determination of CPPs (Critical Process Parameters) and PARs (Proven Acceptable Ranges). A high throughout analytical method to measure N-linked glycans and a Chemometric method to calibrate the results were developed in order to overcome the analytical bottleneck. These studies improved the process understanding, explored the multivariate interactions, and established the relationship between CPPs and CQAs (Critical Quality Attributes). The outcome of the studies was used to develop the integrated control strategy for supporting GMP manufacturing. This approach has been successfully implemented on multiple late stage pipeline molecules including one recently approved monoclonal antibody. In this presentation, the experimental design and relevant statistical analysis methods will be presented using various case studies

Enabling pandemic speed to clinic for SARS-CoV2 neutralizing antibodies

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Influence of genetic factors on the productivity of recombinant Chinese hamster ovary (CHO) cells

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. In this work, influence of some genetic factors on the productivity of recombinant Chinese hamster ovary (CHO) cells has been investigated using a number of experimental techniques. The first system that we have studied is a CHO cell line producing Hepatitis B surface antigen (HbsAg). We have determined the effect of cloned gene dosage on cell growth and HbsAg synthesis and secretion. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (DHFR) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autoradiography and densitometry also gave the same trends. Flow cytometry experiments also show that the amplified clones exhibit a great deal of heterogeneity in single-cell DHFR content as compared to parental cells which were quite homogeneous with respect to intracellular DHFR content. Batch culture experiments carried out in T flasks show that specific growth rates decrease with gene copy number. Secreted HbsAg titers and specific HbsAg secretion rates were found to increase with gene copy number. Specific glucose and glutamine consumption rates as well as specific lactate and NH3 production rates did not vary significantly between the clones although the 1 [...] clone which has the highest relative gene copy number among the clones we studied, exhibited metabolic rates that were slightly higher than those for the other clones. Using pulse-chase experiments we found that efficiency of HbsAg secretion (fraction of total initial HbsAg (0 h chase) that is secreted into the extracellular medium during a 23.5 h chase) decreases and overall efficiency of HbsAg expression (relative amount of HbsAg secreted into the medium, [AU.mm/[...] cells]/relative gene copy number) decreases whereas intracellular HbsAg degradation increases with cloned gene dosage. Comparisons between the relative gene copy number, relative mRNA level, relative amount of total initial HbsAg (0 h chase) and HbsAg secreted into the medium during the longest chase time of 23.5 h for various clones indicate that neither transcription nor translation is limiting at high cloned gene dosage. Our results show that some step after translation limits the overall productivity of these cells at high cloned gene dosage. [...] NMR spectroscopy was carried out using a novel continuous flow packed bed reactor configuration to study the intracellular metabolism of recombinant CHO cells. NMR measurements of CHO cells growing in macroporous collagen microspheres indicate that intracellular phosphorylated metabolite levels including NTP is higher in cells producing HbsAg. These transformed cells producing HbsAg, which exhibit lower specific growth rate in the packed bed, may be utilizing these higher NTP amounts in the HbsAg secretory pathway instead of the cellular biosynthetic pathway. The second system studied here is a CHO cell line producing secreted tissue plasminogen activator (tPA). ATP limitations may influence productivity of secreted cloned proteins in CHO cells and expression of Vitreoscilla hemoglobin (VHb) has been suggested to increase ATP production efficiency in Escherichia coli. Based on these hypotheses, we have cloned the Vitreoscilla hemoglobin gene in CHO cells producing tPA (CHO-tPA) and obtained inducible intracellular expression of VHb in these transfected cells (VHb-CHO). We confirmed the presence of the VHb gene in the genomic DNA of these cells by Southern blots while Western blots were used to demonstrate the expression of VHb protein upon induction with dexamethasone. Batch culture experiments were carried out in order to determine the effect of VHb expression on growth and tPA secretion. We have shown that VHb-CHO cells have a lower specific growth rate compared to CHO-tPA cells but exhibit a significantly higher specific tPA production rate throughout the batch. We have also investigated the effect of some growth factors on cell growth and monoclonal antibody productivity in batch hybridoma cultures. Growth factor addition did not affect specific growth rate of the cells significantly, but significant variation in monoclonal antibody production occurred depending on the growth factor present in the medium. These results indicate that growth factors affect some step in the monoclonal antibody synthesis and secretion pathway without significantly influencing the cell specific growth rate

A Metabolomics Approach to Increasing Chinese Hamster Ovary (CHO) Cell Productivity

Much progress has been made in improving the viable cell density of bioreactor cultures in monoclonal antibody production from Chinese hamster ovary (CHO) cells; however, specific productivity (qP) has not been increased to the same degree. In this work, we analyzed a library of 24 antibody-expressing CHO cell clones to identify metabolites that positively associate with qP and could be used for clone selection or medium supplementation. An initial library of 12 clones, each producing one of two antibodies, was analyzed using untargeted LC-MS experiments. Metabolic model-based annotation followed by correlation analysis detected 73 metabolites that significantly correlated with growth, qP, or both. Of these, metabolites in the alanine, aspartate, and glutamate metabolism pathway, and the TCA cycle showed the strongest association with qP. To evaluate whether these metabolites could be used as indicators to identify clones with potential for high productivity, we performed targeted LC-MS experiments on a second library of 12 clones expressing a third antibody. These experiments found that aspartate and cystine were positively correlated with qP, confirming the results from untargeted analysis. To investigate whether qP correlated metabolites reflected endogenous metabolic activity beneficial for productivity, several of these metabolites were tested as medium additives during cell culture. Medium supplementation with citrate improved qP by up to 490% and more than doubled the titer. Together, these studies demonstrate the potential for using metabolomics to discover novel metabolite additives that yield higher volumetric productivity in biologics production processes