Cell culture quality control by rapid isoenzymatic characterization

Summary Procedures that involve cell cultures require careful quality control to avoid inter- and intraspecies contamination. We have developed and electrophoresis technique that can be used routinely in cell culture laboratories to monitor cell line integrity. The method involves the isoenzymatic separation of nine polymorphic enzymes, three of which can be used for cell line species determinations and seven of which can be used for human cell line characterizations. Examples of how the system has been applied to both inter- and intraspecies identifications are described. The routine application of this protocol would be a valuable asset for laboratories concerned with establishing effective cell culture quality control. This work was supported by Contract N01-CP-9-1003 from the National Cancer Institute, Bethesda, MD.     

A continuous alveolar macrophage cell line: Comparisons with freshly derived alveolar macrophages

Summary Responses of a recently developed rat alveolar macrophage cell (NR8383.1) line were compared to those of freshly derived alveolar macrophages in vitro. Marked inter- and intraspecies heterogeneity in levels of phagocytosis of unopsonizedPseudomonas aeruginosa or zymosan was noted among freshly derived alveolar macrophages from rats, rabbits, and baboons. In contrast, phagocytic responses of alveolar macrophage cell line were predictable and highly reproducible. Similar results were obtained in measuring oxidative burst, as indicated by the production of H₂O₂ and luminol-enhanced chemiluminescence. Responses were again highly variable in freshly derived alveolar macrophages stimulated with zymosan or phorbol myristic acetate; moreover, freshly derived alveolar macrophages exhibited a wide range of chemiluminescence activity in unstimulated cultures. Results strongly suggest that data derived from the continuous alveolar macrophage culture NR8383.1 can be extrapolated to freshly derived alveolar macrophages of various species, and in many experiments will be useful in avoiding the significant animal-to-animal variance observed among freshly derived cell preparations. This work was supported in part by grant A119811 and SCOR HL23578, from the National Institutes of Health, Bethesda, MD. Portions of these studies appeared as a poster presentation at the American Society for Microbiology, Atlanta, GA, 1987.     

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.     

Responses of human peripheral arteries to agonists: similarities and differences

A review of the literature indicates that the effects of many vasoactive substances, and particularly alpha agonists, have been investigated in a variety of human peripheral arterial models, including isolated digital, umbilical, uterine, and mesenteric arteries. The limited comparative data on different human arteries, or on animal vs. human arteries, make definition of inter- and intraspecies differences incomplete at this stage. Examination of recent data from our laboratory using the cystic artery indicates that observed differences in responsiveness of the same artery from different subjects may result from a variety of factors not usually considered in animal models. Hence, age of subjects must be controlled, because factors such as vessel wall thickness, optimal resting tension, and blood pressure vary with age. Even when age is controlled, mildly hypertensive or normotensive females with a family history of hypertension are found to have increased vascular responsiveness to some agonists, which implies the need also to control for sex of the subject. The use of appropriate controls and fastidious laboratory techniques are essential to clearly define inter- and intraspecies similarities and differences.     

Comparability of automated human induced pluripotent stem cell culture: a pilot study

Consistent and robust manufacturing is essential for the translation of cell therapies, and the utilisation automation throughout the manufacturing process may allow for improvements in quality control, scalability, reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently, the effects of manual centrifugation and automated non-centrifugation process steps, performed using TAP Biosystems’ CompacT SelecT automated cell culture platform, upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study, has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However, non-centrifugation hiPSC populations exhibited greater cell yields, greater aggregate rates, increased pluripotency marker expression, and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures, and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories.     

Cell culture monitoring via an auto‐sampler and an integrated multi‐functional off‐line analyzer

Mammalian cell‐based bioprocesses are used extensively for production of therapeutic proteins. Off‐line monitoring of such cultivations via manual sampling is often labor‐intensive and can introduce operator‐dependent error into the process. An integrated multi‐functional off‐line analyzer, the BioProfile FLEX (NOVA Biomedical, Waltham MA) has been developed, which combines the functionality of three off‐line analyzers (a cell counter, an osmometer, and a gas/electrolyte & nutrient/metabolite bio‐profile analyzer) into one device. In addition, a novel automated sampling system has also been developed that allows the BioProfile FLEX to automatically analyze the culture conditions in as many as ten bioreactors. This is the first report on the development and function of this integrated analyzer and an auto‐sampler prototype for monitoring of mammalian cell cultures. Evaluation of the BioProfile FLEX was conducted in two separate laboratories and involved two BioProfile FLEX analyzers and two sets of reference analyzers (Nova BioProfile 400, Beckman‐Coulter Vi‐Cell AS, and Advanced Instruments Osmometer 3900), 13 CHO cell lines and over 20 operators. In general, BioProfile FLEX measurements were equivalent to those obtained using reference analyzers, and the auto‐sampler did not alter the samples it provided to the BioProfile FLEX. These results suggest that the system has the potential to dramatically reduce the manual labor involved in monitoring mammalian cell bioprocesses without altering the quality of the data obtained, and integration with a bioreactor control system will allow feedback control of parameters previously available only for off‐line monitoring. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Organization and evolution of the alcohol dehydrogenase gene in Drosophila

The alcohol dehydrogenase (Adh) gene was isolated from Drosophila simulans and D. mauritiana, and the DNA sequence of a 4.6-kb region, containing the structural gene and flanking sequence, was determined for each. These sequences were compared with the Adh region of D. melanogaster to characterize changes that occur in the Drosophila genome during evolution and to identify conserved sequences of functional importance. Drosophila simulans and D. mauritiana Adh are organized in a manner similar to that of D. melanogaster Adh, including the presence of two promoters for the single Adh gene. This study identified conserved flanking elements that, in conjunction with other studies, suggest regions that may be involved in the control of Adh expression. Inter- and intraspecies comparisons revealed differences in the kinds of sequence changes that have accumulated. Sequence divergence in and around the Adh gene was used to assess inter- and intraspecies evolutionary relationships. Finally, there appears to be an unrelated structural gene located directly 3' of the Adh transcribed region.      

Determination of taxonomic resolution capacity of conventional one-dimensional SDS-polyacrylamide gel electrophoresis of whole-cell proteins using Enterobacteriaceae

The capacity of one-dimensional SDS-PAGE of whole bacterial cells to both separate and cluster taxonomic units is studied using members of Enterobacteriaceae as test material. The results show that intraspecies variation can be detected and on the other hand the degree of taxonomic divergence which still can be grouped together is determined. In addition the system has high tolerance to changes in cell culture conditions making the usage of SDS-PAGE suitable for applications where rapid and reliable bacterial identification is needed.     

Development of at‐line assay to monitor charge variants of MAbs during production

One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro‐heterogeneity can result from post‐translational modifications such as sialylation, galactosylation, C‐terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on‐line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post‐translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on‐line or at‐line measurements of these attributes. In this work, we describe the development of an at‐line assay to separate MAb charge variants in near real‐time, which could ultimately be used to implement on‐line quality control strategies for MAb production. The assay consists of a 2D‐HPLC method with sequential in‐line Protein A and WCX‐10 HPLC column steps. To perform the 2D‐HPLC assay at‐line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D‐HPLC for analysis. With this at‐line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:249–255, 2014     

Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.     

Intraspecies identification of cultured murine cells by using H-2 antigen typing

17 mouse cell lines have been screened with specific sera against H-2 antigens. All the cell lines tested expressed H-2 antigens characteristic of the donor haplotype. The data obtained indicate that H-2 typing of cultured mouse cells can be used as an approach to control their intraspecies diversity.     

Chromosome instability in Spodoptera frugiperda Sf-9 cell line

Homogeneous cell lines are essential in industry and research if reliable and reproducible data are to be obtained. The Spodoptera frugiperda (Sf-9) cell line routinely used for the production of recombinant proteins was found to be heterogeneous, containing a mixture of diploid and tetraploid cells. Using dilution-cloning techniques, diploid and tetraploid subpopulations were isolated from a Sf-9 parental cell line, and their cytogenetic state was monitored using Vinblastine to arrest cells in mitosis. Flow cytometry was used to obtain a snapshot of the predominant subpopulations present to verify the karyological results. The rate at which clonal populations digress into the heterogeneous state was found to be more rapid for the diploid subpopulation, with the emergence of tetraploid cells after only 11 passages, than for the tetraploid subpopulation, where diploid clones appeared after 18 passages. The chromosomes in both diploid and tetraploid subpopulations as well as the parental cell line were found to spontaneously fragment during growth and expansion processes, giving rise to variable chromosome numbers. DNA analysis of cell lines obtained from laboratories worldwide have shown that the Sf-9 cell line used for the production of many recombinant proteins is cytologically unstable, leading to varying degrees of polyploidal state depending on its culture history and supplier.     

Bcl-2 over-expression reduced the serum dependency and improved the nutrient metabolism in a NS0 cells culture

The over-expression of Bcl-2 has greatly improved the culture period, specific growth rate, and maximum viable cell density of NS0 cells culture under low serum condition. Further analysis of these data suggests that a saturation model of the Monod type can be used to represent the relationships of specific growth rate and initial serum concentration. The μ_max andK _s for the Bcl-2 cell line is 0.927 day⁻¹ and 0.947% (v/v) respectively, which are 21% greater and 7% lower respectively than its control counterpart. Study on the amino acid supplementation revealed that Bcl-2 cell lines possess greater improvement in the specific growth rate and maximum viable cell density compared to the control cell lines. A further increase in the amino acid supplementation has resulted a 17% decrease in specific growth rate and no improvement in maximum viable cell density in the control culture. However, the Bcl-2 cell line exhibited a better growth characteristic in this culture condition compared to that of control cell lines. The higher specific growth rate and maximum viable cell density of the Bcl-2 cell line in medium fortified with serum and MEM EAA suggested a more efficient nutrient metabolism compared to that in the control cell line. The low serum and amino acid utilisation rate and the higher cell yield may prove to be important in the development of serum/protein free culture.     

Examining the genetic variation of reference microbial cultures used within food and environmental laboratories using fluorescent amplified fragment length polymorphism analysis

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to genetically fingerprint 'working culture control strains' used by accredited food microbiology laboratories. A working culture control strain is defined as a subculture from a strain initially obtained from an authenticated source [such as the National Collection of Type Cultures (NCTC)] that is maintained for use with routine testing within the laboratory. Working culture control strains from eight food examination laboratories, representing four bacterial species, were analysed by FAFLP; these were Salmonella Nottingham, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus. The resultant FAFLP profiles of the eight working culture control strains for each of these species were compared against the appropriate freeze-dried ampoules obtained directly from NCTC. FAFLP results demonstrated that within 50% of working cultures analysed, several laboratories were routinely using working cultures that were genetically different from the original reference NCTC strains. This study highlights the need for laboratories to review the protocols used to process and maintain control strains and working cultures, with a potential view to utilize single-use quality control materials.     

Automated dynamic fed‐batch process and media optimization for high productivity cell culture process development

Current industry practices for large‐scale mammalian cell cultures typically employ a standard platform fed‐batch process with fixed volume bolus feeding. Although widely used, these processes are unable to respond to actual nutrient consumption demands from the culture, which can result in accumulation of by‐products and depletion of certain nutrients. This work demonstrates the application of a fully automated cell culture control, monitoring, and data processing system to achieve significant productivity improvement via dynamic feeding and media optimization. Two distinct feeding algorithms were used to dynamically alter feed rates. The first method is based upon on‐line capacitance measurements where cultures were fed based on growth and nutrient consumption rates estimated from integrated capacitance. The second method is based upon automated glucose measurements obtained from the Nova Bioprofile FLEX® autosampler where cultures were fed to maintain a target glucose level which in turn maintained other nutrients based on a stoichiometric ratio. All of the calculations were done automatically through in‐house integration with a Delta V process control system. Through both media and feed strategy optimization, a titer increase from the original platform titer of 5 to 6.3 g/L was achieved for cell line A, and a substantial titer increase of 4 to over 9 g/L was achieved for cell line B with comparable product quality. Glucose was found to be the best feed indicator, but not all cell lines benefited from dynamic feeding and optimized feed media was critical to process improvement. Our work demonstrated that dynamic feeding has the ability to automatically adjust feed rates according to culture behavior, and that the advantage can be best realized during early and rapid process development stages where different cell lines or large changes in culture conditions might lead to dramatically different nutrient demands. Biotechnol. Bioeng. 2013; 110: 191–205. © 2012 Wiley Periodicals, Inc.     

A novel scale-down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM)

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 10⁷ cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale.     

Rapid detection of low‐level HeLa cell contamination in cell culture using nested PCR

HeLa cells are a commonly used cell line in many biological research areas. They are not picky for culture medium and proliferate rapidly. HeLa cells are a notorious source of cell cross‐contamination and have been found to be able to contaminate a wide range of cell lines in cell culture. In this study, we reported a simple and efficient method for detecting the presence of HeLa cell contamination in cell culture. HPV‐18 was used as a biomarker. The cell culture supernatant was used directly as the template for nested PCR without extracting nucleic acid. By PCR amplification of the cell culture supernatant with the designed primers, we were able to detect the presence of HeLa cells in the culture. The sensitivity of this method can reach 1%, which is 10‐fold higher than Short tandem repeat sequence (STR) profiling. This simple, rapid, and “noninvasive” quality checking method should find applications in routine cell culture practice.     

Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C

A recently recognized human rhinovirus species C (HRV-C) is associated with up to half of HRV infections in young children. Here we propagated two HRV-C isolates ex vivo in organ culture of nasal epithelial cells, sequenced a new C15 isolate and developed the first, to our knowledge, reverse genetics system for HRV-C. Using contact points for the known HRV receptors, intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptor (LDLR), inter- and intraspecies footprint analyses predicted a unique cell attachment site for HRV-Cs. Antibodies directed to binding sites for HRV-A and -B failed to inhibit HRV-C attachment, consistent with the alternative receptor footprint. HRV-A and HRV-B infected HeLa and WisL cells but HRV-C did not. However, HRV-C RNA synthesized in vitro and transfected into both cell types resulted in cytopathic effect and recovery of functional virus, indicating that the viral attachment mechanism is a primary distinguishing feature of HRV-C.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Biochemical differentiation of a murine ganglioneuroblastoma in tissue culture

A cell line derived from a murine ganglioneuroblastoma has been established in tissue culture. The highly refractile cells have round bodies and develop small processes. Their division time is 36 h under the conditions used. When the cells are grown to high densities, the acetyl cholinesterase activity increases four-fold, and is not affected by nerve growth factor. The behavior of this cell line was compared with C-1300, a murine neuroblastoma that has been cultured in many laboratories. These studies reveal three common features of the two tumors: (1) morphologic appearance in vitro does not correlate with appearance in vivo; (2) both tumors retain the ability to differentiate under appropriate conditions; and (3) biochemical differentiation can be expressed independent of morphology.