Cell Specific Productivity Research Articles

Combined gene and environmental engineering offers a synergetic strategy to enhance r‐protein production in Chinese hamster ovary cells

Environmental growth-inhibition conditions (GICs) have been used extensively for increasing cell-specific productivity (qP ) of Chinese hamster ovary (CHO) cells, with the most common being temperature downshift and sodium butyrate (NaBu) treatment. B lymphocyte-induced maturation protein-1 (BLIMP1) overexpression in CHO cells can also inhibit cell growth and increase product titers and qP . Given the similar responses, this study evaluated the individual and combined effects of BLIMP1 expression, low temperature, and NaBu treatment on culture performance, cell metabolism, and recombinant protein production of CHO cells. As expected, all three interventions decreased cell growth, arrested cells in G1/G0 cell cycle phase, and increased qP . However, CHO cells presented different responses when considering cell viability, recombinant gene expression, and cell metabolism that indicated differences in the molecular loci by which BLIMP1 and GICs generated higher productivities. Combinations of BLIMP1 expression and GICs acted synergistically to inhibit cell growth and maximize r-protein production, with the BLIMP1/NaBu condition leading to the most significant improvements in product titers and qP . This latter condition also proved to substantially increase product yields (up to 9.8 g immunoglobulin G1 [IgG1]/L and 2.2 g erythropoietin-Fc [EPO-Fc]/L) and qP (up to 179 pg/cell/day [pcd] for IgG1 and 30 pcd for EPO-Fc) in high-density perfusion cultures. These findings offered mechanistic insights about the productivity-enhancing effects of BLIMP1 and GICs, as well as their complementarity for generating highly productive processes.

Compartment-specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell-specific productivity for IgG1 producing CHO cells after MTA treatment.

Increasing cell‐specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5′‐deoxy‐5′‐(methylthio)adenosine (MTA), a chemical degradation product of S‐(5′‐adenosyl)‐ʟ‐methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1‐producing CHO cells by 50%. Compartment‐specific 13C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell‐cycle arrest and increased cell volumes. Carbon fluxes into the pentose‐phosphate pathway increased 22 fold in MTA‐treated cells compared to that in non‐MTA‐treated reference cells. Most likely, cytosolic ATP inhibition of phosphofructokinase mediated the carbon detour. Mitochondrial shuttle activity of the α‐ketoglurarate/malate antiporter (OGC) reversed, reducing cytosolic malate transport. In summary, NADPH supply in MTA‐treated cells improved three fold compared to that in non‐MTA‐treated cells, which can be regarded as a major factor for explaining the boosted CSPs.

Exploring the limits of conventional small-scale CHO fed-batch for accelerated on demand monoclonal antibody production

In the field of therapeutic antibody production, diversification of fed-batch strategies is flourishing in response to the market demand. All manufacturing approaches tend to follow the generally accepted dogma of increasing titer since it directly increases manufacturing output. While titer is influenced by the biomass (expressed as IVCD), the culture time and the cell-specific productivity (qP), we changed independently each of these parameters to tune our process strategy towards adapted solutions to individual manufacturing needs. To do so, we worked separately on the increase of the IVCD as high seeding fed-batch capacity. Yet, as intensified fed-batch may not always be possible due to limited facility operational mode, we also separately increased the qP with the addition of specific media additives. Both strategies improved titer by 100% in 14 days relative to the standard fed-batch process with moderate and acceptable changes in product quality attributes. Since intensified fed-batch could rival the cell-specific productivity of a conventional fed-batch, we developed novel hybrid strategies to either allow for acceptable seeding densities without compromising productivity, or alternatively, to push the productivity the furthest in order to reduce timelines.

Monte Carlo-Based Error Propagation for a More Reliable Regression Analysis across Specific Rates in Bioprocesses.

During process development, bioprocess data need to be converted into applicable knowledge. Therefore, it is crucial to evaluate the obtained data under the usage of transparent and reliable data reduction and correlation techniques. Within this contribution, we show a generic Monte Carlo error propagation and regression approach applied to two different, industrially relevant cultivation processes. Based on measurement uncertainties, errors for cell-specific growth, uptake, and production rates were determined across an evaluation chain, with interlinked inputs and outputs. These uncertainties were subsequently included in regression analysis to derive the covariance of the regression coefficients and the confidence bounds for prediction. The usefulness of the approach is shown within two case studies, based on the relations across biomass-specific rate control limits to guarantee high productivities in E. coli, and low lactate formation in a CHO cell fed-batch could be established. Besides the possibility to determine realistic errors on the evaluated process data, the presented approach helps to differentiate between reliable and unreliable correlations and prevents the wrong interpretations of relations based on uncertain data.

Compartment-specific metabolome labeling enables the identification of subcellular fluxes that may serve as promising metabolic engineering targets in CHO cells

13C labeling data are used to calculate quantitative intracellular flux patterns reflecting in vivo conditions. Given that approaches for compartment-specific metabolomics exist, the benefits they offer compared to conventional non-compartmented 13C flux studies remain to be determined. Using compartment-specific labeling information of IgG1-producing Chinese hamster ovary cells, this study investigated differences of flux patterns exploiting and ignoring metabolic labeling data of cytosol and mitochondria. Although cellular analysis provided good estimates for the majority of intracellular fluxes, half of the mitochondrial transporters, and NADH and ATP balances, severe differences were found for some reactions. Accurate flux estimations of almost all iso-enzymes heavily depended on the sub-cellular labeling information. Furthermore, key discrepancies were found for the mitochondrial carriers vAGC1 (Aspartate/Glutamate antiporter), vDIC (Malate/H+ symporter), and vOGC (α-ketoglutarate/malate antiporter). Special emphasis is given to the flux of cytosolic malic enzyme (vME): it could not be estimated without the compartment-specific malate labeling information. Interesting enough, cytosolic malic enzyme is an important metabolic engineering target for improving cell-specific IgG1 productivity. Hence, compartment-specific 13C labeling analysis serves as prerequisite for related metabolic engineering studies.

High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells.

CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs—miRNAs—is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.

Overexpression of transcription factor BLIMP1/prdm1 leads to growth inhibition and enhanced secretory capacity in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells present inherent limitations for processing and secretion of large amounts of recombinant proteins, especially for those requiring complex post-translational processing. To tackle these limitations, we engineered CHO host cells (CHOK1 and CHOS) to overexpress the transcription factor BLIMP1/prdm1 (a master regulator of the highly-secreting phenotype of antibody-producing plasma cells), generating novel CHO cell lines (referred to as CHOB). The CHOB cell lines exhibited decreased cell densities, prolonged stationary phase and arrested cell cycle in G1/G0 phase but simultaneously had significantly greater product titre for recombinant IgG1 (> 2-fold increase) coupled with a significantly greater cell-specific productivities (> 3-fold increase). We demonstrated that the improved productive phenotype of CHOB cells resulted from a series of changes to cell physiology and metabolism. CHOB cells showed a significantly greater ER size and increased protein synthesis and secretion capacity compared to control cells. In addition, CHOB cells presented a metabolic profile that favoured energy production to support increased recombinant protein production. This study indicated that a cell engineering approach based on BLIMP1 expression offers great potential for improving the secretory capacity of CHO cell hosts utilised for manufacture of recombinant biopharmaceuticals. Our findings also provides a greater understanding of the relationship between cell growth and productivity, valuable generic information for improving productive phenotypes for CHO cell lines during industrial cell line development.

Selection of CHO host and recombinant cell pools by inhibition of the proteasome results in enhanced product yields and cell specific productivity

Chinese hamster ovary (CHO) cells are the leading mammalian cell expression platform for biotherapeutic recombinant molecules yet some proteins remain difficult to express (DTE) in this, and other, systems. In recombinant cell lines expressing DTE proteins, cellular processes to restore proteostasis can be triggered when the folding and modification capabilities are exceeded, including the unfolded protein response and ER-associated degradation (ERAD) and proteasomal degradation. We therefore investigated whether the proteasome activity of CHO cells was linked to their ability to produce recombinant proteins. We found cell lines with diverse monoclonal antibody (mAb) productivity show different susceptibilities to inhibitors of proteasome activity. Subsequently, we applied selective pressure using proteasome inhibitors on mAb producing cells to determine the impact on cell growth and recombinant protein production, and to apply proteasome selective pressure above that of a metabolic selection marker during recombinant cell pool construction. The presence of proteasome inhibitors during cell pool construction expressing two different model molecules, including a DTE Fc-fusion protein, resulted in the generation of cell pools with enhanced productivity. The increased productivities, and ability to select for higher producing cells, has potential to improve clonal selection during upstream processes of DTE proteins.

Considerations of the Impacts of Cell-Specific Growth and Production Rate on Clone Selection—A Simulation Study

For the manufacturing of complex biopharmaceuticals using bioreactors with cultivated mammalian cells, high product concentration is an important objective. The phenotype of the cells in a reactor plays an important role. Are clonal cell populations showing high cell-specific growth rates more favorable than cell lines with higher cell-specific productivities or vice versa? Five clonal Chinese hamster ovary cell populations were analyzed based on the data of a 3-month-stability study. We adapted a mechanistic cell culture model to the experimental data of one such clonally derived cell population. Uncertainties and prior knowledge concerning model parameters were considered using Bayesian parameter estimations. This model was used then to define an inoculum train protocol. Based on this, we subsequently simulated the impacts of differences in growth rates (±10%) and production rates (±10% and ±50%) on the overall cultivation time, including making the inoculum train cultures; the final production phase, the volumetric titer in that bioreactor and the ratio of both, defined as overall process productivity. We showed thus unequivocally that growth rates have a higher impact (up to three times) on overall process productivity and for product output per year, whereas cells with higher productivity can potentially generate higher product concentrations in the production vessel.

STEP® vectors for rapid generation of stable transfected CHO cell pools and clones with high expression levels and product quality homogeneity of difficult-to-express proteins

Many proteins produced in CHO cells need evaluation for their clinical and commercial potential. Traditional methods based on stable clone generation are slow and unsuitable for screening larger numbers of proteins, while transient expression technologies are fast but unpredictable regarding product quality and lacking an optional path to subcloning. The STEP® vector technology introduced here combines the best properties of both methods. STEP® vectors contain a strong transcriptional cassette driving expression of a bicistronic mRNA. The gene-of-interest (GOI) is cloned upstream of a functionally impaired zeocin resistance gene (FI-Zeo) whose translation is coupled to that of the GOI through an IRES. Stable transfected cells surviving zeocin selection produce high levels of FI-Zeo and thus, high levels of the GOI-encoded protein. By using different spacers, the translational coupling efficiency and selection strength can be controlled allowing maximization of expression of any GOI. Production of laronidase and factor VII (FVII) is presented as examples of unrelated, difficult-to-express (DTE) proteins. First step is rapid generation of transfected pools with the STEP® vectors. All high expressing surviving pools showed high product quality homogeneity as did monoclonal cell lines obtained from the top pools. Up to 500 μg/mL laronidase was obtained with virtually identical glycosylation profile as reference product. For FVII, cell specific productivity of 0.45 pg/cell/day with 50 IU/μg protein matched highest reported levels of reference product even before process development. Taken together, STEP® vector technology is ideally suited for rapid, small to large-scale production of DTE proteins compared to traditional methods.

Bioreactor production of rVSV-based vectors in Vero cell suspension cultures.

The Vero cell line is the most used continuous cell line in viral vaccine manufacturing. This adherent cell culture platform requires the use of surfaces to support cell growth, typically roller bottles, or microcarriers. We have recently compared the production of rVSV‐ZEBOV on Vero cells between microcarrier and fixed‐bed bioreactors. However, suspension cultures are considered superior with regard to process scalability. Therefore, we further explore the Vero suspension system for recombinant vesicular stomatitis virus (rVSV)‐vectored vaccine production. Previously, this suspension cell line was only able to be cultivated in a proprietary medium. Here, we expand the adaptation and bioreactor cultivation to a serum‐free commercial medium. Following small‐scale optimization and screening studies, we demonstrate bioreactor productions of highly relevant vaccines and vaccine candidates against Ebola virus disease, HIV, and coronavirus disease 2019 in the Vero suspension system. rVSV‐ZEBOV, rVSV‐HIV, and rVSVInd‐msp‐SF‐Gtc can replicate to high titers in the bioreactor, reaching 3.87 × 107 TCID50/ml, 2.12 × 107 TCID50/ml, and 3.59 × 109 TCID50/ml, respectively. Furthermore, we compare cell‐specific productivities, and the quality of the produced viruses by determining the ratio of total viral particles to infectious viral particles.

Increasing lipid yield in Yarrowia lipolytica through phosphoketolase and phosphotransacetylase expression in a phosphofructokinase deletion strain

BackgroundLipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism.ResultsBuilding on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively.ConclusionsYields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production.

Transfection of miR-31* boosts oxidative phosphorylation metabolism in the mitochondria and enhances recombinant protein production in Chinese hamster ovary cells

MicroRNAs are increasingly being used to enhance relevant pathways of interest during CHO cell line development and to optimise biopharmaceutical production processes. Previous studies have demonstrated that genetic manipulation of microRNAs has led to the development of highly productive phenotypes by increasing cell density through modifying the cell cycle, extending the culture lifespan by delaying apoptotic mechanisms, or improving the energetic flux by targeting mitochondrial metabolism. Re-programming mitochondrial metabolism has arisen as a potential area of interest due to the potential to decrease the Warburg effect and increase cell specific productivity with significant impact on the manufacture of recombinant therapeutic proteins. In this study, we have demonstrated a role for miR-31* to enhance specific productivity in CHO cells by boosting oxidative phosphorylation in the mitochondria. A detailed analysis of the mitochondrial metabolism revealed that miR-31* transfection increases basal oxygen consumption and spare respiratory capacity that leads to an increase in ATP production. Additionally, a proteomic analysis unveiled a number of potential targets involved in fatty acid metabolism and the TCA cycle, both implicated in mitochondrial metabolism. This data demonstrates a potential role for miR-31* to reprogramme the mitochondrial energetic metabolism and increase recombinant protein production in CHO cells.

4-(2,5-Dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide improves monoclonal antibody production in a Chinese hamster ovary cell culture.

There is a continuous demand to improve monoclonal antibody production for medication supply and medical cost reduction. For over 20 years, recombinant Chinese hamster ovary cells have been used as a host in monoclonal antibody production due to robustness, high productivity and ability to produce proteins with ideal glycans. Chemical compounds, such as dimethyl sulfoxide, lithium chloride, and butyric acid, have been shown to improve monoclonal antibody production in mammalian cell cultures. In this study, we aimed to discover new chemical compounds that can improve cell-specific antibody production in recombinant Chinese hamster ovary cells. Out of the 23,227 chemicals screened in this study, 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide was found to increase monoclonal antibody production. The compound suppressed cell growth and increased both cell-specific glucose uptake rate and the amount of intracellular adenosine triphosphate during monoclonal antibody production. In addition, the compound also suppressed the galactosylation on a monoclonal antibody, which is a critical quality attribute of therapeutic monoclonal antibodies. Therefore, the compound might also be used to control the level of the galactosylation for the N-linked glycans. Further, the structure-activity relationship study revealed that 2,5-dimethylpyrrole was the most effective partial structure of 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide on monoclonal antibody production. Further structural optimization of 2,5-dimethylpyrrole derivatives could lead to improved production and quality control of monoclonal antibodies.

Effect of East Asian atmospheric particulate matter deposition on bacterial activity and community structure in the oligotrophic Northwest Pacific

Large amounts of anthropogenic East Asian (EA) particulate matters (PM), containing inorganic nutrients and organic matter, are deposited in the oligotrophic Northwest Pacific Ocean. However, the effects of such deposition on marine microbes remain unclear. In this study, the effect of EA PM deposition on marine bacteria was assessed by five on-board microcosm experiments, conducted in oligotrophic basins of the South China Sea. The addition of EA PM to the sampling water induced a clear shift in bacterial community composition from prevailing oligotrophs (i.e., SAR 11 clade, Prochlorococcus, AEGEAN-169 marine group) to less common copiotrophs (i.e., Alteromonas, Ruegeria, Flavobacteriaceae) and thus a slight increase in bacterial diversity. The shift to more active community composition, as well as stimulation of PM nutrients, resulted in a large increase in cell-specific and bulk bacterial production. In contrast, there were only minor changes in bacterial abundance, possibly due to increased top-down mortality. The EA PM also exhibited a stronge toxic effect on pico-cyanobacteria, leading to a significant decrease in their proportion. Moreover, the responses of bacterial metabolism and community composition exhibited significant relationships with the hydrographic condition of the locations. Stronger promotion effects of the EA PM on bacterial production and community shift from oligotrophs to copiotrophs was demonstrated at the more oligotrophic sites with lower chlorophyll a concentrations. These results suggest that PM deposition from polluted areas has the potential to alter the typical oligotrophic microbiomes and change the net metabolic balance of the bacterial community. These will then influence the dynamics of carbon flow in microbial food webs and biogeochemical cycles, especially with the trend of global warming and expansion of low-chlorophyll regions.

Autophagy-inducing peptide increases CHO cell monoclonal antibody production in batch and fed-batch cultures.

The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy ("self-eating") is a cellular process that allows cells to mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy-inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed-batch serum-free cultures producing a human Immunoglobulin G1 (IgG1). Interestingly, the addition of 1-4 µM AIP enhanced productivity in a concentration-dependent manner. Cell-specific productivity increased up to 1.8-fold in batch cultures, while in fed-batch cultures a maximum twofold increase in IgG concentration was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields.

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth.Key points• Less and more efficient glucose utilization for suspension cell growth• Concerted alteration of metabolic enzyme activity and protein expression• Protein candidates to interfere glycolytic activity in MDCK cells

Understanding the effect of increased cell specific productivity on galactosylation of monoclonal antibodies produced using Chinese hamster ovary cells

Achieving optimal productivity and desired product quality of the therapeutic monoclonal antibody (mAb) is one of the primary goals of process development. Across the various mAb programs at our company, we observed that increasing the specific productivity (qp) results in a decrease in the % galactosylation (%Gal) level on the protein. In order to gain further insight into this correlation, cells were cultured under different process conditions such as pH or media osmolality or in the presence of supplements such as sodium butyrate. A range of qp and N-glycan profiles were obtained with the greatest changes observed under high pH (lower qp, higher %Gal), higher osmolality (higher qp, lower %Gal) or sodium butyrate (moderately higher qp, moderately lower %Gal) conditions. Abundance of individual glycan species highlighted different bottlenecks in the N-glycosylation pathway depending on the treatment condition. Transcriptomics analysis was performed to identify changes in gene expression profiles that correlate with the inverse relationship between qp and %Gal. Results showed downregulation of Beta-1,4-galactosyltransferase 1 (B4GalT1), UDP-GlcNAc and Mn2+ transporter (slc35a3 and slc39a8 respectively) for the high osmolality conditions. Significant downregulation of slc39a8 (Mn2+ transporter) was observed for the sodium butyrate condition. No significant differences were observed for any of the genes in the N-glycosylation pathway under the high pH condition even though this condition showed highest %Gal. Together, data suggests that different treatments have distinct complex mechanisms by which the overall glycan levels of a mAb are influenced. Further studies based on these results will help build the knowledge necessary to design strategies to obtain the desired productivity and product quality of mAbs.

Monitoring cell productivity for the production of recombinant proteins by flow cytometry: An effective application using the cold capture assay.

Due to the increasing economic and social relevance of biotherapeutics, their production processes are continually being reconsidered and reoptimized in an effort to secure higher product concentrations and qualities. Monitoring the productivity of cultured cells is therefore a critically important part of the cultivation process. Traditionally, this is achieved by determining the overall product titer by high performance liquid chromatography (HPLC), and then calculating the specific cell productivity based on this titer and an associated viable cell density. Unfortunately, this process is typically time‐consuming and laborious. In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively time‐effective, and potentially cheaper alternative for monitoring the productivity of a cell culture.

Medullary stromal cells synergize their production and capture of CCL21 for T-cell emigration from neonatal mouse thymus.

The release of newly selected αβT cells from the thymus is key in establishing a functional adaptive immune system. Emigration of the first cohorts of αβT cells produced during the neonatal period is of particular importance, because it initiates formation of the peripheral αβT-cell pool and provides immune protection early in life. Despite this, the cellular and molecular mechanisms of thymus emigration are poorly understood. We examined the involvement of diverse stromal subsets and individual chemokine ligands in this process. First, we demonstrated functional dichotomy in the requirement for CCR7 ligands and identified CCL21, but not CCL19, as an important regulator of neonatal thymus emigration. To explain this ligand-specific requirement, we examined sites of CCL21 production and action and found Ccl21 gene expression and CCL21 protein distribution occurred within anatomically distinct thymic areas. Although Ccl21 transcription was limited to subsets of medullary epithelium, CCL21 protein was captured by mesenchymal stroma consisting of integrin α7+ pericytes and CD34+ adventitial cells at sites of thymic exit. This chemokine compartmentalization involved the heparan sulfate-dependent presentation of CCL21 via its C-terminal extension, explaining the absence of a requirement for CCL19, which lacks this domain and failed to be captured by thymic stroma. Collectively, we identified an important role for CCL21 in neonatal thymus emigration, revealing the importance of this chemokine in initial formation of the peripheral immune system. Moreover, we identified an intrathymic mechanism involving cell-specific production and presentation of CCL21, which demonstrated a functional synergy between thymic epithelial and mesenchymal cells for αβT-cell emigration.