Ellen McCormick

A single nutrient feed supports both chemically defined NS0 and CHO fed‐batch processes: Improved productivity and lactate metabolism

A chemically defined nutrient feed (CDF) coupled with basal medium preloading was developed to replace a hydrolysate‐containing feed (HCF) for a fed‐batch NS0 process. The CDF not only enabled a completely chemically defined process but also increased recombinant monoclonal antibody titer by 115%. Subsequent tests of CDF in a CHO process indicated that it could also replace the hydrolysate‐containing nutrient feed in this expression system as well as providing an 80% increase in product titer. In both CDF NS0 and CHO processes, the peak lactate concentrations were lower and, more interestingly, lactate metabolism shifted markedly from net production to net consumption when cells transitioned from exponential to stationary growth phase. Subsequent investigations of the lactate metabolic shift in the CHO CDF process were carried out to identify the cause(s) of the metabolic shift. These investigations revealed several metabolic features of the CHO cell line that we studied. First, glucose consumption and lactate consumption are strictly complementary to each other. The combined cell specific glucose and lactate consumption rate was a constant across exponential and stationary growth phases. Second, Lactate dehydrogenase (LDH) activity fluctuated during the fed‐batch process. LDH activity was at the lowest when lactate concentration started to decrease. Third, a steep cross plasma membrane glucose gradient exists. Intracellular glucose concentration was more than two orders of magnitude lower than that in the medium. Fourth, a large quantity of citrate was diverted out of mitochondria to the medium, suggesting a partially truncated tricarboxylic acid (TCA) cycle in CHO cells. Finally, other intermediates in or linked to the glycolytic pathway and the TCA cycle, which include alanine, citrate, isocitrate, and succinate, demonstrated a metabolic shift similar to that of lactate. Interestingly, all these metabolites are either in or linked to the pathway downstream of pyruvate, but upstream of fumarate in glucose metabolism. Although the specific mechanisms for the metabolic shift of lactate and other metabolites remain to be elucidated, the increased understanding of the metabolism of CHO cultures could lead to future improvements in medium and process development.

A predictive high‐throughput scale‐down model of monoclonal antibody production in CHO cells

Multi‐factorial experimentation is essential in understanding the link between mammalian cell culture conditions and the glycoprotein product of any biomanufacturing process. This understanding is increasingly demanded as bioprocess development is influenced by the Quality by Design paradigm. We have developed a system that allows hundreds of micro‐bioreactors to be run in parallel under controlled conditions, enabling factorial experiments of much larger scope than is possible with traditional systems. A high‐throughput analytics workflow was also developed using commercially available instruments to obtain product quality information for each cell culture condition. The micro‐bioreactor system was tested by executing a factorial experiment varying four process parameters: pH, dissolved oxygen, feed supplement rate, and reduced glutathione level. A total of 180 micro‐bioreactors were run for 2 weeks during this DOE experiment to assess this scaled down micro‐bioreactor system as a high‐throughput tool for process development. Online measurements of pH, dissolved oxygen, and optical density were complemented by offline measurements of glucose, viability, titer, and product quality. Model accuracy was assessed by regressing the micro‐bioreactor results with those obtained in conventional 3 L bioreactors. Excellent agreement was observed between the micro‐bioreactor and the bench‐top bioreactor. The micro‐bioreactor results were further analyzed to link parameter manipulations to process outcomes via leverage plots, and to examine the interactions between process parameters. The results show that feed supplement rate has a significant effect (P < 0.05) on all performance metrics with higher feed rates resulting in greater cell mass and product titer. Culture pH impacted terminal integrated viable cell concentration, titer and intact immunoglobulin G titer, with better results obtained at the lower pH set point. The results demonstrate that a micro‐scale system can be an excellent model of larger scale systems, while providing data sets broader and deeper than are available by traditional methods. Biotechnol. Bioeng. 2009; 104: 1107–1120.

Engineering specificity of starter unit selection by the erythromycin‐producing polyketide synthase

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase‐like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ‐containing loading modules are shown here to contain a key arginine residue at their active site. Site‐specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13‐methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex‐tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1‐TE, in which the 4′‐phosphopante‐theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro‐ducts in detectable amounts.

Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367.

Enhanced productivity of NS0 cells in fed-batch culture with cholesterol nanoparticle supplementation

NS0 cells are an important industrial cell line for the production of therapeutic monoclonal antibodies. Culturing these cells is challenging because they are cholesterol auxotrophs, and providing cholesterol to the cells is hampered by the low solubility of lipids in aqueous medium. Limited loading capacity, precipitation, instability, and toxicity are associated with traditional delivery methods that involve solvents or carrier molecules. In this work, nanoparticle cholesterol mixtures (NCM) were produced by electrohydrodynamic spraying and added directly to a cholesterol auxotroph NS0 cell line. Compared to a cholesterol‐cyclodextrin solution and a commercial proprietary cholesterol solution, SyntheChol™ NS0 supplement, NCM is significantly less cytotoxic. In the fed batch cell culture process, product titer was increased by 32% when the NCM supplement replaced SyntheChol™ NS0 supplement. An even greater product titer improvement, 64%, was achieved when both NCM and SyntheChol™ NS0 supplements were used in the fed‐batch process.

Dynamic Ranking of Clones with Process Conditions Using a High-Throughput Micro-bioreactor Platform

The economic production of recombinant proteins in mammalian cells is highly dependent upon the selection of high-producing cell lines (Rathore and Winkle Nat Biotechnol 27(1):26–34, 2009). Traditional screening technologies remain limited in their ability to mimic the bioreactor environment where the production clone will ultimately need to perform. Because of its inherent throughput, the SimCell platform uniquely enables the application of a Dynamic Ranking of Clones selection strategy. This approach achieves a comprehensive ranking of multiple cell line performances as a function of both media and process variables in a single experiment. In this study, eight CHO clones producing a recombinant monoclonal antibody were screened across several process variations, including different feeding strategies, temperature shifts and pH control profiles. A total of 240 micro-bioreactors were run in a single experiment, for two weeks, to assess the scale-down model as a high-throughput tool for clone evaluation. Clones were ranked for growth and titer performance in the fed-batch experimental design. Best and worst performers were clearly identified. The second phase experiment utilized 180 micro-bioreactors in a full factorial design comprised of a subset of 12 clone/process combinations run in parallel in duplicate shake flasks. Good correlation between the micro-bioreactor predictions and those made in shake flasks was obtained (R 2 = 0.90).