Development of a novel capillary electrophoresis method for quantitative measurements of intracellular recombinant protein titer.

The measurement of volumetric titer is an integral step in the assessment and selection of a production cell line and cell culture process. The production of monoclonal antibodies (mAbs), a major class of therapeutic proteins, in Chinese Hamster Ovary (CHO) cell lines is challenging due to the clone-to-clone variations in the intrinsic capability to secrete a biologically complex protein. The measurement of intracellular mAb concentration could be a valuable tool to determine the ratio of intracellular to secreted product and be part of the evaluation of potential mAb productive cell lines. High throughput automation is a valuable tool that is used in bioprocess development to reduce work intensive steps. When coupled with the Simple Western (Wes) platform, automated capillary electrophoresis is an efficient method to measure recombinant protein concentration. In this study, we demonstrate the utility of using the automated Wes to rapidly measure intracellular titer and then compare the intracellular titer, volumetric titer and specific productivity between high and low production CHO clones expressing a model human IgG1 mAb.

In silico design of CMV promoter binding oligonucleotides and their impact on inhibition of gene expression in Chinese hamster ovary cells.

Modulation of expression levels of endogenous or recombinant genes can be of great interest for diverse applications, such as the study of genotype-phenotype relationships for a gene of interest, or fine-tuning of transcription to determine physiologically relevant effects of gene expression levels. During the last decades, several synthetic biology tools were established to control gene expression in mammalian cells such as Chinese hamster ovary (CHO) cells, one of the most important cell systems for basic research as well as the production of biopharmaceuticals. Here we describe the use of triplex forming oligos (TFOs), short RNA or ssDNA molecules that can bind to the major grove of their target duplex with great specificity, to control transgene expression in CHO cells. For proof of concept, a panel of TFOs with a size of 10-20 nts were designed with the help of the on-line tool Triplexator targeting the viral cytomegalovirus (CMV) promoter/enhancer region controlling the downstream reporter gene hCD4. The effect of TFOs was tested as ssDNA oligos pre-annealed to the promoter/enhancer region in vitro as well as upon endogenous transcription of the TFO as an RNA molecule binding to their target duplex in vivo. Results showed that not only binding of the TFO, but the exact location of triplex formation within the promoter/enhancer is paramount for transcription inhibition. After relieving a binding conflict by introducing a point mutation within the CMV promoter, longer TFOs (26-30 nts) could be designed and analysed. Selected TFOs achieved a reduction in recombinant hCD4 expression of up to 85% in CHO-K1 cells.

Characterization of cell cycle and apoptosis in Chinese hamster ovary cell culture using flow cytometry for bioprocess monitoring.

Chinese hamster ovary (CHO) cells are by far the most important mammalian cell lines used for producing antibodies and other therapeutic proteins. It is critical to fully understand their physiological conditions during a bioprocess in order to achieve the highest productivity and the desired product quality. Flow cytometry technology possesses unique advantages for measuring multiple cellular attributes for a given cell and examining changes in cell culture heterogeneity over time that can be used as metrics for enhanced process understanding and control strategy. Flow cytometry-based assays were utilized to examine the progression of cell cycle and apoptosis in three case studies using different antibody-producing CHO cell lines in both fed-batch and perfusion bioprocesses. In our case studies, we found that G0/G1 phase distribution and early apoptosis accumulation responded to subtle changes in culture conditions, such as pH shifting or momentary glucose depletion. In a perfusion process, flow cytometry provided an insightful understanding of the cell physiological status under a hypothermic condition. More importantly, these changes in cell cycle and apoptosis were not detected by a routine trypan blue exclusion-based cell counting and viability measurement. In summary, integration of flow cytometry into bioprocesses as a process analytical technology tool can be beneficial for establishing optimum process conditions and process control.

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy.

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.

An Integrated Solution-Based Rapid Sample Preparation Procedure for the Analysis of N-Glycans From Therapeutic Monoclonal Antibodies.

Consistent glycosylation in therapeutic monoclonal antibodies is a major concern in the biopharmaceutical industry as it impacts the drug's safety and efficacy and manufacturing processes. Large numbers of samples are created for the analysis of glycans during various stages of recombinant proteins drug development. Profiling and quantifying protein N-glycosylation is important but extremely challenging due to its microheterogeneity and more importantly the limitations of existing time-consuming sample preparation methods. Thus, a quantitative method with fast sample preparation is crucial for understanding, controlling, and modifying the glycoform variance in therapeutic monoclonal antibody development. Presented here is a rapid and highly quantitative method for the analysis of N-glycans from monoclonal antibodies. The method comprises a simple and fast solution-based sample preparation method that uses nontoxic reducing reagents for direct labeling of N-glycans. The complete work flow for the preparation of fluorescently labeled N-glycans takes a total of 3 h with less than 30 min needed for the release of N-glycans from monoclonal antibody samples.

Selection of CHO host cell subclones with increased specific antibody production rates by repeated cycles of transient transfection and cell sorting.

Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by "hard fact" gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO-K1 and CHO-S host cells that possess an improved cellular machinery for transient antibody production. The long-term goal of this study was both to identify host cells that yield recombinant cell lines with on average higher productivity, but also to study the molecular differences that characterize such cells, independent of the site of gene integration or gene amplification. To identify such cells we optimized the procedure for transient transfection by electroporation to a degree that gave uniform transfer of plasmid DNA into nearly 100% of the cells and resulted in reproducible average productivities, with a standard deviation of 16% between independent experiments. Using this optimized protocol, the 1% of cells with the highest specific productivity was sorted and subcloned with a cold capture secretion assay. Upon re-transfection, the resulting subclones showed the same specific productivity as their respective parental cell line. To enrich for cells with potentially stable improved properties, the 1% highest producers were sorted three times, 2 days after transient transfection each, and the enriched population was again sorted into microtiter plates for subcloning. For each of the two parental cell lines tested, three subclones were obtained that had a threefold higher specific productivity after transient transfection. This property was stable for approximately 3 months, indicating that the changes in productivity were regulatory and not mutational.

A study on the temperature dependency and time course of the cold capture antibody secretion assay.

The cold capture assay as described by Brezinsky et al. [Brezinsky, S.C.G., Chiang, G.G., Szilvasi, A., Mohan, S., Shapiro, R.I., MacLean, A., Sisk, W., Thill, G., 2003. A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J. Immunol. Methods 277, 141-155] stands out as the most simple of single cell secretion assays which can be used to sort for high productivity in recombinant cell lines. At low temperatures the process of protein release from transport vesicles is assumed to be delayed as both vesicle fusion and product release is slowed, so that secreted proteins can be stained on the cell surface using a fluorescent antibody. Typically, the fluorescent signal obtained correlates to the cell specific production rate of the analysed cell. In the present study we compared staining of human antibody producing CHO cells performed at different temperatures and we observed the fluorescent signal over 24h. We found that the staining temperature did not influence signal intensity. The fluorescent signal was stable for 24h at 4 degrees C, decreased to 80% at room temperature (21 degrees C), while it decreased significantly already after 2h at 37 degrees C. Initially, the fluorescent signal was observed on the cell surface, however, at later stages it was found in compartments in the cytoplasm. Finally we compared differences in signal stability depending on whether the antibody used for staining bound to the light or heavy chain of the product and on whether the fluorescent label was a relatively stable protein (phycoerythrin) or a pH-dependent small molecule (FITC). Our results indicate that the secreted product is trapped by the staining antibody on the cell surface at all temperatures. Subsequently these aggregates are endocytosed by the cells, a process which is slowed down at low temperatures.