Cell culture process operations for recombinant protein production.

The market for protein therapeutics has grown significantly over the past two decades and the pace of development continues to increase. It is a challenge to the industry to maintain the desired quality attributes while accelerating delivery to patients, reducing the cost of goods, and providing production flexibility. Efficient manufacturing scale production of protein therapeutics is required to continue to meet the needs of the patients and stockholders. This chapter describes batch, fed-batch, and perfusion processes and their utilization in the production of monoclonal antibodies and other therapeutic proteins. In addition, we have provided detailed discussions of the ongoing challenges of lactate metabolism and the future prospects of process monitoring and control.

NMR-based metabolomics of mammalian cell and tissue cultures.

NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

Defining process design space for monoclonal antibody cell culture.

The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified.

Dexamethasone effects on rat hepatocyte spheroid formation and function.

Hepatocytes cultured on moderately adhesive surfaces or in spinner flasks spontaneously self-assemble into spherical tissue-like aggregates (spheroids). These spheroids have smooth surfaces and tissue-like polarized cell morphology, including bile canalicular-like channels, and maintain high viability and liver-specific functions for extended culture periods. Dexamethasone (DEX), a synthetic glucocorticoid, is known to elicit various responses in gene expression, and is often added to hepatocyte culture medium. The morphology and liver-specific protein production of hepatocyte spheroids were assessed under DEX concentrations ranging from 50 nM to 10 microM. DEX altered the kinetics of spheroid formation in a concentration-dependent fashion, with increasing concentrations inhibiting aggregation and promoting aggregate disassembly on culture dishes. DEX addition to spinner cultures resulted in smaller, more irregularly shaped spheroids and a higher incidence of aggregate clumping. Albumin and urea production were also higher in DEX cultures, but this effect was not as sensitive to concentration and occurred irrespective of the state of aggregation. RTPCR was utilized to assess the mRNA levels of extracellular matrix proteins, E-cadherin, and cytochrome P-450 enzymes. Results indicated a slight increase in fibronectin and collagen III mRNA early in the cultures, possibly contributing to the changes in morphology observed.

Characterization of a hollow fiber bioartificial liver device.

A three-compartment bioartificial liver (BAL) has been developed for potential treatment of fulminant hepatic failure. It has been shown previously that viability and liver-specific functions were maintained in laboratory-scale bioreactors of such design. In this study, the performance of hepatocytes in a clinical-scale bioartificial liver was verified by sustained specific production rates of albumin and urea, along with oxygen consumption rates for up to 56 h and liver-specific gene expression for up to 72 h. In addition, transmission of porcine endogenous retrovirus and other type C retroviral particles across the hollow fibers was not detected under both normal and extreme operating fluxes. These results demonstrate that the clinical-scale BAL performs at a level similar to the laboratory scale and that it offers a viral barrier against porcine retroviruses.

Three-dimensional co-culture of hepatocytes and stellate cells.

Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.

Structural polarity and functional bile canaliculi in rat hepatocyte spheroids.

Primary hepatocytes self-assemble into spheroids that possess tight junctions and microvilli-lined channels. We hypothesized that polarity develops gradually and that the channels structurally and functionally resemble bile canaliculi. Immunofluorescence labeling of apical and basolateral proteins demonstrated reorganization of the membrane proteins into a polarized distribution during spheroid culture. By means of fluorescent dextran diffusion and confocal microscopy, an extensive network of channels was revealed in the interior of the spheroids. These channels connected over several planes and opened to pores on the surface. To examine the content of apical proteins in the channel membranes, the bile canalicular enzyme dipeptidyl peptidase IV (DPPIV) was localized using a fluorogenic substrate, Ala-Pro-cresyl violet. The results show that DPPIV activity is heterogeneously distributed in spheroids and localized in part to channels. Bile acid excretion was then investigated to demonstrate functional polarity. A fluorescent bile acid analogue, fluorescein isothiocyanate-labeled glycocholate, was taken up into the spheroids and excreted into bile canalicular channels. Due to the structural polarity of spheroids and their ability to excrete bile into channels, they are a unique three-dimensional model of in vitro liver tissue self-assembly. (Videoanimations of some results are available at http://hugroup.cems.umn.edu/research_movies).