Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability, process validation, and scale-up for manufacturing.

Biochemical and functional testing of a humanized monoclonal antibody directed against Respiratory Syncytial Virus (Synagis) has been performed to evaluate cell line stability, support process validation, and to demonstrate "comparability" during the course of process development. Using a variety of analytical methods, product manufactured at different sites and in bioreactors from 20 litres to 10,000 litres was shown to be biochemically and functionally equivalent. The biochemical testing for microheterogeneity found on Synagis included evaluation of changes in post-translational modifications such as deamidation, truncation, and carbohydrate structure. Studies were also performed to support cell line stability assessment and cell culture process validation. Cell culture conditions were deliberately varied in an attempt to determine if this would have an impact on the microheterogeneity of the product. In these studies Synagis was produced from cells cultured beyond the population doublings achieved at the maximum manufacturing scale, under conditions of low glucose, and using harvest times outside of the historical manufacturing operating range. Results showed that there was a different pattern of glycosylation during the early stages of bioreactor culture. No other changes in microheterogeneity were apparent for the other culture conditions studied. In summary, comparability assessment demonstrated that the Synagis manufacturing process is robust and consistent resulting in a predictable and reproducible monoclonal antibody product.     

Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number of approved biotherapeutics increases.     

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.     

Towards robust cell culture processes — Unraveling the impact of media preparation by spectroscopic online monitoring

Biopharmaceutical manufacturing processes can be affected by variability in cell culture media, e.g. caused by raw material impurities. Although efforts have been made in industry and academia to characterize cell culture media and raw materials with advanced analytics, the process of industrial cell culture media preparation itself has not been reported so far. Within this publication, we first compare mid‐infrared and two‐dimensional fluorescence spectroscopy with respect to their suitability as online monitoring tools during cell culture media preparation, followed by a thorough assessment of the impact of preparation parameters on media quality. Through the application of spectroscopic methods, we can show that media variability and its corresponding root cause can be detected online during the preparation process. This methodology is a powerful tool to avoid batch failure and is a valuable technology for media troubleshooting activities. Moreover, in a design of experiments approach, including additional liquid chromatography–mass spectrometry analytics, it is shown that variable preparation parameters such as temperature, power input and preparation time can have a strong impact on the physico‐chemical composition of the media. The effect on cell culture process performance and product quality in subsequent fed‐batch processes was also investigated. The presented results reveal the need for online spectroscopic methods during the preparation process and show that media variability can already be introduced by variation in media preparation parameters, with a potential impact on scale‐up to a commercial manufacturing process.     

A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying

Freeze-drying of bacterial cells with retained viability and activity after storage requires appropriate formulation, i.e. mixing of physiologically adapted cell populations with suitable protective agents, and control of the freeze-drying process. Product manufacturing may alter the clinical effects of probiotics and it is essential to identify and understand possible factor co-dependencies during manufacturing. The physical solid-state behavior of the formulation and the freeze-drying parameters are critical for bacterial survival and thus process optimization is important, independent of strain. However, the maximum yield achievable is also strain-specific and strain survival is governed by e.g. medium, cell type, physiological state, excipients used, and process. The use of preferred compatible solutes for cross-protection of Lactobacilli during industrial manufacturing may be a natural step to introduce robustness, but knowledge is lacking on how compatible solutes, such as betaine, influence formulation properties and cell survival. This study characterized betaine formulations, with and without sucrose, and tested these with the model lactic acid bacteria Lactobacillus coryniformis Si3. Betaine alone did not act as a lyo-protectant and thus betaine import prior to freeze-drying should be avoided. Differences in protective agents were analyzed by calorimetry, which proved to be a suitable tool for evaluating the characteristics of the freeze-dried end products.     

Application of process quality engineering techniques to improve the understanding of the in vitro processing of stem cells for therapeutic use

The translation of experimental cell-based therapies to volume produced commercially successful clinical products requires the development of capable, economic, scaleable (and therefore frequently necessarily automated) manufacturing processes. Application of proven quality engineering techniques will be required to interrogate, optimise, and control in vitro cell culture processes to regulatory and clinically acceptable specifications. We have used a Six Sigma inspired quality engineering approach to design and conduct a factorial screening experiment to investigate the expansion process of a population of primary bone marrow-derived human mesenchymal stem cells on a scaleable automated cell culture platform. Key cell culture process inputs (seeding density, serum concentration, media quantity and incubation time) and important cell culture process responses (cell number and the expression of alkaline phosphatase, STRO-1, CD105 and CD71) were identified as experimental variables. The results rank the culture factors and significant culture factor interactions by the magnitude of their effect on each of the process responses. This level of information is not available from conventional single factor cell culture studies but is essential to efficiently identify sources of variation and foci for further process optimisation. Systematic quality engineering approaches such as those described here will be essential for the design of regulated cell therapy manufacturing processes because of their focus on identifying the sources of and the control of variation, an issue that is at the core of current Good Manufacturing Practice.     

Acquired resistance to tyrosine kinase inhibitors during cancer therapy

Selective tyrosine kinase inhibitors have emerged as important therapeutic agents in the treatment of a variety of human malignancies. Although several of these inhibitors have marked clinical activity, it is widely recognized that the overall value of these agents is substantially limited by the acquisition of drug resistance, which eventually arises in most, if not all treated patients. Mechanisms of drug resistance are beginning to be elucidated through the molecular analysis of clinical specimens as well as through cell culture modeling. By identifying resistance mechanisms, it should be possible to develop 'second-generation' inhibitors as well as rational drug combinations that can overcome or even prevent acquired resistance to kinase inhibitors, thereby enhancing clinical benefit.     

FluBlok,a next generation influenza vaccine manufactured in insect cells

FluBlok, a recombinant trivalent hemagglutinin (rHA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV). Its manufacturing process presents the possibility for safe and expeditious vaccine production. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. The high purity of the antigen enables administration at higher doses without a significant increase in side-effects in human subjects.The insect cell–baculovirus production technology is particularly suitable for influenza where annual adjustment of the vaccine is required. The baculovirus–insect expression system is generally considered a safe production system, with limited growth potential for adventitious agents. Still regulators question and challenge the safety of this novel cell substrate as FluBlok continues to advance toward product approval. This review provides an overview of cell substrate characterization for expresSF cell line used for the manufacturing of FluBlok.In addition, this review includes an update on the clinical development of FluBlok. The highly purified protein vaccine, administered at three times higher antigen content than TIV, is well tolerated and results in stronger immunogenicity, a long lasting immune response and provides cross-protection against drift influenza viruses.     

Longitudinal two-dimensional gas chromatography mass spectrometry as a non-destructive at-line monitoring tool during cell manufacturing identifies volatile features correlative to cell product quality

Background aimsCell therapies have emerged as a potentially transformative therapeutic modality in many chronic and incurable diseases. However, inherent donor and patient variabilities, complex manufacturing processes, lack of well-defined critical quality attributes and unavailability of in-line or at-line process or product analytical technologies result in significant variance in cell product quality and clinical trial outcomes. New approaches for overcoming these challenges are needed to realize the potential of cell therapies.MethodsHere the authors developed an untargeted two-dimensional gas chromatography mass spectrometry (GC×GC-MS)-based method for non-destructive longitudinal at-line monitoring of cells during manufacturing to discover correlative volatile biomarkers of cell proliferation and end product potency.ResultsSpecifically, using mesenchymal stromal cell cultures as a model, the authors demonstrated that GC×GC-MS of the culture medium headspace can effectively discriminate between media types and tissue sources. Headspace GC×GC-MS identified specific volatile compounds that showed a strong correlation with cell expansion and product functionality quantified by indoleamine-2,3-dioxygenase and T-cell proliferation/suppression assays. Additionally, the authors discovered increases in specific volatile metabolites when cells were treated with inflammatory stimulation.ConclusionsThis work establishes GC×GC-MS as an at-line process analytical technology for cell manufacturing that could improve culture robustness and may be used to non-destructively monitor culture state and correlate with end product function.     

Advances in primary recovery: centrifugation and membrane technology

Significant and continual improvements in upstream processing for biologics have resulted in challenges for downstream processing, both primary recovery and purification. Given the high cell densities achievable in both microbial and mammalian cell culture processes, primary recovery can be a significant bottleneck in both clinical and commercial manufacturing. The combination of increased product titer and low viability leads to significant relative increases in the levels of process impurities such as lipids, intracellular proteins and nucleic acid versus the product. In addition, cell culture media components such as soy and yeast hydrolysates have been widely applied to achieve the cell culture densities needed for higher titers. Many of the process impurities can be negatively charged at harvest pH and can form colloids during the cell culture and harvest processes. The wide size distribution of these particles and the potential for additional particles to be generated by shear forces within a centrifuge may result in insufficient clarification to prevent fouling of subsequent filters. The other residual process impurities can lead to precipitation and increased turbidity during processing and even interference with the performance of the capturing chromatographic step. Primary recovery also poses significant challenges owing to the necessity to execute in an expedient manner to minimize both product degradation and bioburden concerns. Both microfiltration and centrifugation coupled with depth filtration have been employed successfully as primary recovery processing steps. Advances in the design and application of membrane technology for microfiltration and dead-end filtration have contributed to significant improvements in process performance and integration, in some cases allowing for a combination of multiple unit operations in a given step. Although these advances have increased productivity and reliability, the net result is that optimization of primary recovery processes has become substantially more complicated. Ironically, the application of classical chemical engineering approaches to overcome issues in primary recovery and purification (e.g., turbidity and trace impurity removal) are just recently gaining attention. Some of these techniques (e.g., membrane cascades, pretreatment, precipitation, and the use of affinity tags) are now seen almost as disruptive technologies. This paper will review the current and potential future state of research on primary recovery, including relevant papers presented at the 234th American Chemical Society (ACS) National Meeting in Boston.     

Case study: The characterization and implementation of dielectric spectroscopy (biocapacitance) for process control in a commercial GMP CHO manufacturing process

Dielectric spectroscopy (biocapacitance) is an up-and-coming technology for real time monitoring of biomass in cell culture processes and has opened the door for next-generation cell culture process control techniques such as automated on-demand nutrient feeding. In this case study we empirically demonstrate the lower limit of quantitation (LOQ), probe-to-probe consistency, and scalability of in situ biocapacitance probes using data generated from small- and large-scale Chinese hamster ovary (CHO) bioreactor cultures. The process understanding experiments culminated in the use of biocapacitance for process control in the current good manufacturing practices (GMP) manufacturing environment, first to automate the dilution of seed train cultures during scale-up stages and later as a method of predicting future glucose demand. The automated biomass-probe-based inoculation strategy yielded consistent results in six consecutive seed trains in the GMP manufacturing suite. In the process of improving our understanding of the technology we determined that biocapacitance could additionally be used as an indicator of a shift in the salt balance of a cell culture, and that collecting real time biomass data via biocapacitance has the potential to reduce the total timeline for feed strategy development by providing additional insights into culture performance which are not otherwise apparent using conventional optical cell counting methods.     

Advances in automated cell washing and concentration

The successful commercialization of cell therapies requires thorough planning and consideration of product quality, cost and scale of the manufacturing process. The implementation of automation can be central to a robust and reproducible manufacturing process at industrialized scales. There have been a number of wash-and-concentrate devices developed for cell manufacturing. These technologies have arisen from transfusion medicine, hematopoietic stem cell and biologics manufacturing where operating mechanisms are distinct from manual centrifugation. This review describes the historical origin and fundamental technologies underlying each currently available wash-and-concentrate device as well as their relative advantages and disadvantages in cell therapy applications. Understanding the specific attributes and limitations of these technologies is essential to optimizing cell therapy manufacturing.     

Demonstration of robust host cell protein clearance in biopharmaceutical downstream processes

Residual host cell protein impurities (HCPs) are a key component of biopharmaceutical process related impurities. These impurities need to be effectively cleared through chromatographic steps in the downstream purification process to produce safe and efficacious protein biopharmaceuticals. A variety of strategies to demonstrate robust host cell protein clearance using scale-down studies are highlighted and compared. A common strategy is the "spiking" approach, which is widely employed in clearance studies for well-defined impurities. For HCPs this approach involves spiking cell culture harvest, which is rich in host cell proteins, into the load material for all chromatographic steps to assess their clearance ability. However, for studying HCP clearance, this approach suffers from the significant disadvantage that the vast majority of host cell protein impurities in a cell culture harvest sample are not relevant for a chromatographic step that is downstream of the capture step in the process. Two alternative strategies are presented here to study HCP clearance such that relevance of those species for a given chromatographic step is taken into consideration. These include a "bypass" strategy, which assumes that some of the load material for a chromatographic step bypasses that step and makes it into the load for the subsequent step. The second is a "worst-case" strategy, which utilizes information obtained from process characterization studies. This involves operating steps at a combination of their operating parameters within operating ranges that yield the poorest clearance of HCPs over that step. The eluate from the worst case run is carried forward to the next chromatographic step to assess its ability to clear HCPs. Both the bypass and worst-case approaches offer significant advantages over the spiking approach with respect to process relevance of the HCP impurity species being studied. A combination of these small-scale validation approaches with large-scale HCP clearance data from clinical manufacturing and manufacturing consistency runs is used to demonstrate robust HCP clearance for the downstream purification process of an Fc fusion protein. The demonstration of robust HCP clearance through this comprehensive strategy can potentially be used to eliminate the need for routine analytical testing or for establishing acceptance criteria for these impurities as well as to demonstrate robust operation of the entire downstream purification process.     

Mechanism of antibody reduction in cell culture production processes

We recently observed a significant disulfide reduction problem during the scale‐up of a manufacturing process for a therapeutic antibody using a CHO expression system. Under certain conditions, extensive reduction of inter‐chain disulfide bonds of an antibody produced by CHO cell culture may occur during the harvest operations and/or the protein A chromatography step, resulting in the observation of antibody fragments (light chain, heavy chain, and various combination of both) in the protein A pools. Although all conditions leading to disulfide reduction have not been completely identified, an excessive amount of mechanical cell lysis generated at the harvest step appears to be an important requirement for antibody reduction (Trexler‐Schmidt et al., 2010 ). We have been able to determine the mechanism by which the antibody is reduced despite the fact that not all requirements for antibody reduction were identified. Here we present data strongly suggesting that the antibody reduction was caused by a thioredoxin system or other reducing enzymes with thioredoxin‐like activity. The intracellular reducing enzymes and their substrates/cofactors apparently were released into the harvest cell culture fluid (HCCF) when cells were exposed to mechanical cell shear during harvest operations. Surprisingly, the reducing activity in the HCCF can last for a long period of time, causing the reduction of inter‐chain disulfide bonds in an antibody. Our findings provide a basis for designing methods to prevent the antibody reduction during the manufacturing process. Biotechnol. Bioeng. 2010;107:622–632. © 2010 Wiley Periodicals, Inc.     

Cost-effective manufacture of an allogeneic GM-CSF-secreting breast tumor vaccine in an academic cGMP facility

BACKGROUND: GM-CSF-secreting, allogeneic cell-based cancer vaccines have shown promise for the treatment of a variety of solid tumors. We have now applied this approach to breast cancer. The aim of these studies was to optimize expansion parameters, qualify the manufacturing process, and establish expected outcomes for cGMP-compliant manufacturing of two GM-CSF-secreting breast tumor cell lines. METHODS: The variables affecting the efficiency of expanding and formulating two allogeneic GM-CSF-secreting cell lines, 2T47D-V and 3SKBR3-7, were systematically evaluated. Production criteria investigated included alternative cell culture vessels (flasks vs. cell factories), centrifugation time and speed variables for large volume cell concentration, cell seeding density, the minimal concentration of FBS required for maximal cell expansion, and the dose and timing of irradiation in relation to cryopreservation. RESULTS: These studies demonstrate that, in comparison with standard 150-cm2 tissue culture flasks, Nunc 10-Stack Cell Factories are a more efficient and practical cell culture vessel for vaccine cell line manufacture. Centrifugation optimization studies using the COBE 2991 Cell Processor established that a speed of 2000 r.p.m. (450 g) for 2 min reliably concentrated the cells while maintaining acceptable viability and bioactivity. Radiation studies established that lethal irradiation prior to cryopreservation does not compromise the quality of the product, as measured by post-thaw cell viability and GM-CSF cell line-specific secretion levels. Finally, studies aimed at optimizing the production of one vaccine cell line, 3SKBR3-7, demonstrated that seeding the cells at a higher density and maintaining them in half the initial concentration of FBS maximized the yield of bioactive cells, resulting in significant cost savings. DISCUSSION: A manufacturing process that simultaneously maximizes cell yield, minimizes cell manipulation and maintains vaccine cell potency is critical for producing cell-based cancer vaccines in an academic setting. These studies define a feasible, reproducible and cost-effective methodology for production of a GM-CSF-secreting breast cancer vaccine that is cGMP compliant.     

Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures

Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost‐effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale‐down (USD) mimics requiring 25–110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost‐effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario.     

Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

N‐linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N‐linked glycosylation patterns in a CHO‐S cell fed‐batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High‐throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N‐linked glycosylation patterns during a fed‐batch process as a function of time as well as the manipulated variables. A constant and varying mAb N‐linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model‐based evaluation of feeding regimes using high‐throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1135–1148, 2016     

A hybrid mechanistic-empirical model for in silico mammalian cell bioprocess simulation

In cell culture processes cell growth and metabolism drive changes in the chemical environment of the culture. These environmental changes elicit reactor control actions, cell growth response, and are sensed by cell signaling pathways that influence metabolism. The interplay of these forces shapes the culture dynamics through different stages of cell cultivation and the outcome greatly affects process productivity, product quality, and robustness. Developing a systems model that describes the interactions of those major players in the cell culture system can lead to better process understanding and enhance process robustness. Here we report the construction of a hybrid mechanistic-empirical bioprocess model which integrates a mechanistic metabolic model with subcomponent models for cell growth, signaling regulation, and the bioreactor environment for in silico exploration of process scenarios. Model parameters were optimized by fitting to a dataset of cell culture manufacturing process which exhibits variability in metabolism and productivity. The model fitting process was broken into multiple steps to mitigate the substantial numerical challenges related to the first-principles model components. The optimized model captured the dynamics of metabolism and the variability of the process runs with different kinetic profiles and productivity. The variability of the process was attributed in part to the metabolic state of cell inoculum. The model was then used to identify potential mitigation strategies to reduce process variability by altering the initial process conditions as well as to explore the effect of changing CO₂ removal capacity in different bioreactor scales on process performance. By incorporating a mechanistic model of cell metabolism and appropriately fitting it to a large dataset, the hybrid model can describe the different metabolic phases in culture and the variability in manufacturing runs. This approach of employing a hybrid model has the potential to greatly facilitate process development and reactor scaling.     

Simultaneous assessment of migration and proliferation of murine fibrosarcoma cells, as affected by hydroxyurea, vinblastine, cytochalasin B, Razoxane and interferon

Abstract. Using porous cell culture chambers, we have simultaneously assessed growth and locomotion of cancer cells to investigate whether certain agents affect cell motility in addition to cell division. First, cells from a murine fibrosarcoma cell line, 1.0/L1, were grown in ordinary flask cultures to determine appropriate cell inocula. Doses of agents were selected to reduce the final 4 day culture cellularity to about 50%, when present during the last two days of culturing. Secondly, the effects of these agents on cell numbers in the porous chambers and on cell migration out of the chambers ('emigration fraction') were recorded. We also examined, using a similar type of porous chamber, whether the agents could affect leucocyte chemotaxis. Hydroxyurea (an inhibitor of DNA synthesis) reduced cancer cell emigration as well as cell growth, without interfering with leucocyte chemotaxis. Cytochalasin B (a microfilament disrupting agent) inhibited cancer cell motility and growth, as well as leucocyte chemotaxis. Vinblastine (a microtubule disrupting agent), at the very low dose chosen, reduced cancer cell growth, but did not consistently affect the migration of either cell type. The experimental anti-metastasis agent Razoxane reduced growth, but had no detectable effects on motility. High doses of natural murine interferon–α/β weakly inhibited both cancer cell growth and locomotion. This motivates for further studies of these and other cytokines, as treatment with agents inhibiting cancer cell locomotion might possibly prevent peri-operative spread of cancer in patients.     

Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types

Viral contaminations of biopharmaceutical manufacturing cell culture facilities are a significant threat and one for which having a risk mitigation strategy is highly desirable. High temperature, short time (HTST) mammalian cell media treatment may potentially safeguard manufacturing facilities from such contaminations. HTST is thought to inactivate virions by denaturing proteins of the viral capsid, and there is evidence that HTST provides ample virucidal efficacy against nonenveloped or naked viruses such as mouse minute virus (MMV), a parvovirus. The aim of the studies presented herein was to further delineate the susceptibility of MMV, known to have contaminated mammalian cell manufacturing facilities, to heat by exposing virus‐spiked cell culture media to a broad range of temperatures and for various times of exposure. The results of these studies show that HTST is capable of inactivating MMV by three orders of magnitude or more. Thus, we believe that HTST is a useful technology for the purposes of providing a barrier to adventitious contamination of mammalian cell culture processes in the biopharmaceutical industry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009