Matrix attachment regions enhance transgene expression by manipulating position-dependent effects in stably transfected CHO-K1 cells.

We previously found that the position of matrix attachment regions (MARs) within the vector significantly affects its ability to enhance transgenic expression in the recombinant protein production. This study aims to systematically investigate the position-dependent impacts of MAR on transgene expression. We observed a significant increase in enhanced green fluorescent protein (eGFP) expression levels in stably transfected CHO-K1 cells with either MAR 1-68 or MAR X-29 when MARs located upstream of the promoter. This increase was especially evident with MAR flanked the expression cassette. Concurrently, a substantial increase was observed in the percentage of eGFP-expressing cells, with 97.8% and 96.0% in MAR-containing constructs versus 73.7% in MAR-absent constructs. Further analysis of erythropoietin (EPO) expression revealed that constructs with flanking MARs induced the highest EPO productivity. Bioinformatics analysis revealed that certain specific transcription factors are important in modulating the transcription process. In conclusion, vectors harboring both MARs around the expression cassette constitute an optimal construct for enhanced recombinant protein production in CHO-K1 cells. This insight underscores the importance of strategic MAR incorporation in vector design for optimized recombinant protein expression.

Identification and characterization of CHO host-cell proteins in monoclonal antibody bioprocessing.

Host-cell proteins (HCPs) are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.

Kinetic and functional analysis of abundant microRNAs in extracellular vesicles from normal and stressed cultures of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells release and exchange large quantities of extracellular vesicles (EVs). EVs are highly enriched in microRNAs (miRs, or miRNAs), which are responsible for most of their biological effects. We have recently shown that the miR content of CHO EVs varies significantly under culture stress conditions. Here, we provide a novel stoichiometric ("per-EV") quantification of miR and protein levels in large CHO EVs produced under ammonia, lactate, osmotic, and age-related stress. Each stress resulted in distinct EV miR levels, with selective miR loading by parent cells. Our data provide a proof of concept for the use of CHO EV cargo as a diagnostic tool for identifying culture stress. We also tested the impact of three select miRs (let-7a, miR-21, and miR-92a) on CHO cell growth and viability. Let-7a-abundant in CHO EVs from stressed cultures-reduced CHO cell viability, while miR-92a-abundant in CHO EVs from unstressed cultures-promoted cell survival. Overexpression of miR-21 had a slight detrimental impact on CHO cell growth and viability during late exponential-phase culture, an unexpected result based on the reported antiapoptotic role of miR-21 in other mammalian cell lines. These findings provide novel relationships between CHO EV cargo and cell phenotype, suggesting that CHO EVs may exert both pro- and antiapoptotic effects on target cells, depending on the conditions under which they were produced.

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

Genome-wide CRISPR/Cas9 knockout screening to mitigate cell growth inhibition induced by histone deacetylase inhibitors in recombinant CHO cells.

Histone deacetylase inhibitors (iHDACs) have been extensively studied as enhancers of therapeutic protein production in recombinant Chinese hamster ovary (CHO) (rCHO) cell cultures. However, the addition of iHDACs reduces the viable cell concentration (VCC) in rCHO cell cultures, thereby reducing their potential to enhance therapeutic protein production. To mitigate the negative effects of iHDACs on VCC, screening using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based single-gene knockout (KO) library in rCHO cells was performed in the presence of CI994, a member of iHDACs, and 10 potential KO genes that enhanced the VCC of CI994-treated rCHO cells were identified. Among these, Bcor was validated as a promising KO target that improved VCC without negatively affecting the specific productivity in the presence of CI994. Bcor KO increased the VCC and therapeutic protein concentrations in both batch and fed-batch cultures in the presence of CI994. Taken together, these findings highlight the potential of the whole-genome CRISPR/Cas9-based single-gene KO cell library to identify KO target genes for the development of iHDAC-resistant rCHO cells for enhanced therapeutic protein production.

Host cell protein networks as a novel co-elution mechanism during protein A chromatography.

Host cell proteins (HCPs) are process-related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc-based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co-purification of HCPs with the therapeutic protein are either HCP-drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc-based proteins by LC-MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co-eluted with less than three Fc-based proteins indicating a drug-specific co-purification for most HCPs. Only ten HCPs co-purified with over 50% of the 23 Fc-based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co-elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein-protein interaction networks. Here, we propose an additional mechanism for HCP co-elution involving protein-protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies.

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells.

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins.

Exploring metabolic effects of dipeptide feed media on CHO cell cultures by in silico model-guided flux analysis.

There is a growing interest in perfusion or continuous processes to achieve higher productivity of biopharmaceuticals in mammalian cell culture, specifically Chinese hamster ovary (CHO) cells, towards advanced biomanufacturing. These intensified bioprocesses highly require concentrated feed media in order to counteract their dilution effects. However, designing such condensed media formulation poses several challenges, particularly regarding the stability and solubility of specific amino acids. To address the difficulty and complexity in relevant media development, the biopharmaceutical industry has recently suggested forming dipeptides by combining one from problematic amino acids with selected pairs to compensate for limitations. In this study, we combined one of the lead amino acids, L-tyrosine, which is known for its poor solubility in water due to its aromatic ring and hydroxyl group, with glycine as the partner, thus forming glycyl-L-tyrosine (GY) dipeptide. Subsequently, we investigated the utilization of GY dipeptide during fed-batch cultures of IgG-producing CHO cells, by changing its concentrations (0.125 × , 0.25 × , 0.5 × , 1.0 × , and 2.0 ×). Multivariate statistical analysis of culture profiles was then conducted to identify and correlate the most significant nutrients with the production, followed by in silico model-guided analysis to systematically evaluate their effects on the culture performance, and elucidate metabolic states and cellular behaviors. As such, it allowed us to explain how the cells can more efficiently utilize GY dipeptide with respect to the balance of cofactor regeneration and energy distribution for the required biomass and protein synthesis. For example, our analysis results uncovered specific amino acids (Asn and Gln) and the 0.5 × GY dipeptide in the feed medium synergistically alleviated the metabolic bottleneck, resulting in enhanced IgG titer and productivity. In the validation experiments, we tested and observed that lower levels of Asn and Gln led to decreased secretion of toxic metabolites, enhanced longevity, and elevated specific cell growth and titer. KEY POINTS: • Explored the optimal Tyr dipeptide for the enhanced CHO cell culture performance • Systematically analyzed effects of dipeptide media by model-guided approach • Uncovered synergistic metabolic utilization of amino acids with dipeptide.

Enhancing Human Glycoprotein Hormones Production in CHO Cells Using Heterologous Beta-Chain Signal Peptides.

We studied the influence of heterologous signal peptides in the ß-chains of glycoprotein hormones on the biosynthesis of these hormones in a transiently transfected culture of Chinese hamster ovary cells CHO S. When the natural signal peptides of the ß-chains were replaced with the heterologous signal peptide of human serum albumin, cell productivity was increased 2-2.5 times for human luteinizing hormone, human chorionic gonadotropin, and human thyroid-stimulating hormone, but not for human follicle-stimulating hormone. No significant increase in cell productivity was observed for human azurocidin signal peptide and human glycoprotein hormone α-chain signal peptide. The used approach allows quick assessing the effect of heterologous signal peptides on the biosynthesis of heterodimeric proteins of various classes.

CHOGlycoNET: Comprehensive glycosylation reaction network for CHO cells.

Chinese hamster ovary (CHO) cells are extensively used for the production of glycoprotein therapeutics proteins, for which N-linked glycans are a critical quality attribute due to their influence on activity and immunogenicity. Manipulation of protein glycosylation is commonly achieved through cell or process engineering, which are often guided by mathematical models. However, each study considers a unique glycosylation reaction network that is tailored around the cell line and product at hand. Herein, we use 200 glycan datasets for both recombinantly produced and native proteins from different CHO cell lines to reconstruct a comprehensive reaction network, CHOGlycoNET, based on the individual minimal reaction networks describing each dataset. CHOGlycoNET is used to investigate the distribution of mannosidase and glycosyltransferase enzymes in the Golgi apparatus and identify key network reactions using machine learning and dimensionality reduction techniques. CHOGlycoNET can be used for accelerating glycomodel development and predicting the effect of glycoengineering strategies. Finally, CHOGlycoNET is wrapped in a SBML file to be used as a standalone model or in combination with CHO cell genome scale models.

A nutrition algorithm to optimize feed and medium composition using genome-scale metabolic models.

The optimization of animal feeds and cell culture media are problems of interest to a wide range of industries and scientific disciplines. Both problems are dictated by the properties of an organism's metabolism. However, due to the tremendous complexity of metabolic systems, it can be difficult to predict how metabolism will respond to changes in nutrient availability. A common tool used to capture the complexity of metabolism in a computational framework is a genome-scale metabolic model (GEM). GEMs are useful for predicting the fluxes of reactions within an organism's metabolism. To optimize feed or media, in silico experiments can be performed with GEMs by systematically varying nutritional constraints and predicting metabolic activity. In this way, the influence of various nutritional changes on metabolic outcomes can be evaluated. However, this methodology does not guarantee an optimal solution. Here, we develop a nutrition algorithm that utilizes linear programming to search the entire flux solution space of possible dietary intervention strategies to identify the most efficient changes to nutrition for a desirable metabolic outcome. We illustrate the utility of the nutrition algorithm on GEMs of Atlantic salmon (Salmo salar) and Chinese hamster ovary (CHO) cell metabolism and find that the nutrition algorithm makes predictions that not only align with experimental findings but reveal new insights into promising feeding strategies. We show that the nutrition algorithm is highly versatile and customizable to meet the user's needs. For instance, we demonstrate that the nutrition algorithm can be used to predict feed/media compositions that maximize profit margins. While the nutrition algorithm can be used to define an optimal feed/medium ab initio, it can also identify minimal changes to be made to an existing feed/medium to drive the largest metabolic shift. Moreover, the nutrition algorithm can target multiple metabolic pathways simultaneously with only a marginal increase in computational expense. While the nutrition algorithm has its limitations, we believe that this tool can be leveraged in a broad range of biotechnological applications to enhance the feed/medium optimization process.

The secretory pathway - the key for unlocking the potential of Chinese hamster ovary cell factories for manufacturing therapeutic proteins.

Mammalian cell factories (in particular the CHO cell system) have been crucial in the rise of biopharmaceuticals. Mammalian cells have compartmentalized organelles where intricate networks of proteins manufacture highly sophisticated biopharmaceuticals in a specialized production pipeline - the secretory pathway. In the bioproduction context, the secretory pathway functioning is key for the effectiveness of cell factories to manufacture these life-changing medicines. This review describes the molecular components and events involved in the secretory pathway, and provides a comprehensive summary of the intracellular steps limiting the production of therapeutic proteins as well as the achievements in engineering CHO cell secretory machinery. We also consider antibody-producing plasma cells (so called "professional" secretory cells) to explore the mechanisms underpinning their unique secretory function/features. Such understandings offer the potential to further enhancement of the current CHO cell production platforms for manufacturing next generation of biopharmaceuticals.

How reliable are Chinese hamster ovary (CHO) cell genome-scale metabolic models?

Genome-scale metabolic models (GEMs) possess the power to revolutionize bioprocess and cell line engineering workflows thanks to their ability to predict and understand whole-cell metabolism in silico. Despite this potential, it is currently unclear how accurately GEMs can capture both intracellular metabolic states and extracellular phenotypes. Here, we investigate this knowledge gap to determine the reliability of current Chinese hamster ovary (CHO) cell metabolic models. We introduce a new GEM, iCHO2441, and create CHO-S and CHO-K1 specific GEMs. These are compared against iCHO1766, iCHO2048, and iCHO2291. Model predictions are assessed via comparison with experimentally measured growth rates, gene essentialities, amino acid auxotrophies, and 13 C intracellular reaction rates. Our results highlight that all CHO cell models are able to capture extracellular phenotypes and intracellular fluxes, with the updated GEM outperforming the original CHO cell GEM. Cell line-specific models were able to better capture extracellular phenotypes but failed to improve intracellular reaction rate predictions in this case. Ultimately, this work provides an updated CHO cell GEM to the community and lays a foundation for the development and assessment of next-generation flux analysis techniques, highlighting areas for model improvements.

Long term culture promotes changes to growth, gene expression, and metabolism in CHO cells that are independent of production stability.

Phenotypic stability of Chinese hamster ovary (CHO) cells over long term culture (LTC) presents one of the most pressing challenges in the development of therapeutic protein manufacturing processess. However, our current understanding of the consequences of LTC on recombinant (r-) CHO cell lines is still limited, particularly as clonally-derived cell lines present distinct production stability phenotypes. This study evaluated changes of culture performance, global gene expression, and cell metabolism of two clonally-derived CHO cell lines with a stable or unstable phenotype during the LTC (early [EP] vs. late [LP] culture passages). Our findings indicated that LTC altered the behavior of CHO cells in culture, in terms of growth, overall gene expression, and cell metabolism. Regardless whether cells were categorized as stable or unstable in terms of r-protein production, CHO cells at LP presented an earlier decline in cell viability and loss of any observable stationary phase. These changes were parallelled by the upregulation of genes involved in cell proliferation and survival pathways (i.e., MAPK/ERK, PI3K-Akt). Stable and unstable CHO cell lines both showed increased consumption of glucose and amino acids at LP, with a parallel accumulation of greater amounts of lactate and TCA cycle intermediates. In terms of production stability, we found that decreased r-protein production in the unstable cell line directly correlated to the loss in r-gene copy number and r-mRNA expression. Our data revealed that LTC produced ubiquitious effects on CHO cell phenotypes, changes that were rooted in alterations in cell transcriptome and metabolome. Overall, we found that CHO cells adapted their cellular function to proliferation and survival during the LTC, some of these changes may well have limited effects on overall yield or specific productivity of the desired r-product, but they may be critical toward the capacity of cells to handle r-proteins with specific molecular features.

The microRNomes of Chinese hamster ovary (CHO) cells and their extracellular vesicles, and how they respond to osmotic and ammonia stress.

A new area of focus in Chinese hamster ovary (CHO) biotechnology is the role of small (exosomes) and large (microvesicles or microparticles) extracellular vesicles (EVs). CHO cells in culture exchange large quantities of proteins and RNA through these EVs, yet the content and role of these EVs remain elusive. MicroRNAs (miRs or miRNA) are central to adaptive responses to stress and more broadly to changes in culture conditions. Given that EVs are highly enriched in miRs, and that EVs release large quantities of miRs both in vivo and in vitro, EVs and their miR content likely play an important role in adaptive responses. Here we report the miRNA landscape of CHO cells and their EVs under normal culture conditions and under ammonia and osmotic stress. We show that both cells and EVs are highly enriched in five miRs (among over 600 miRs) that make up about half of their total miR content, and that these highly enriched miRs differ significantly between normal and stress culture conditions. Notable is the high enrichment in miR-92a and miR-23a under normal culture conditions, in contrast to the high enrichment in let-7 family miRs (let-7c, let-7b, and let-7a) under both stress conditions. The latter suggests a preserved stress-responsive function of the let-7 miR family, one of the most highly preserved miR families across species, where among other functions, let-7 miRs regulate core oncogenes, which, depending on the biological context, may tip the balance between cell cycle arrest and apoptosis. While the expected-based on their profound enrichment-important role of these highly enriched miRs remains to be dissected, our data and analysis constitute an important resource for exploring the role of miRs in cell adaptation as well as for synthetic applications.

CHOmpact: A reduced metabolic model of Chinese hamster ovary cells with enhanced interpretability.

Metabolic modeling has emerged as a key tool for the characterization of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimize the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges. Genome-scale metabolic models (GeMs) of CHO cells are very large (>6000 reactions) and are difficult to constrain to yield physiologically consistent flux distributions. The large scale of GeMs also makes the interpretation of their outputs difficult. To address these challenges, we have developed CHOmpact, a reduced metabolic network that encompasses 101 metabolites linked through 144 reactions. Our compact reaction network allows us to deploy robust, nonlinear optimization and ensure that the computed flux distributions are physiologically consistent. Furthermore, our CHOmpact model delivers enhanced interpretability of simulation results and has allowed us to identify the mechanisms governing shifts in the anaplerotic consumption of asparagine and glutamate as well as an important mechanism of ammonia detoxification within mitochondria. CHOmpact, thus, addresses key challenges of large-scale metabolic models and will serve as a platform to develop dynamic metabolic models for the control and optimization of biopharmaceutical cell culture processes.

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

Fusion with CTP increases the stability of recombinant neuritin.

Neuritin is a vital neurotrophin that plays an essential role in recovery from nerve injury and neurodegenerative diseases and may become a new target for treating these conditions. However, improving neuritin protein stability is an urgent problem. In this study, to obtain active and stable neuritin proteins, we added a carboxyl-terminal peptide (CTP) sequence containing four O-linked glycosylation sites to the C-terminus of neuritin and cloned it into the Chinese hamster ovary (CHO) expression system. The neuritin-CTP protein was purified using a His-Tag purification strategy after G418 screening of stable high-expression cell lines. Ultimately, we obtained neuritin-CTP protein with a purity >90%. Functional analyses showed that the purified neuritin-CTP protein promoted the neurite outgrowth of PC12 cells, and stability experiments showed that neuritin stability was increased by adding CTP. These results indicate that neuritin protein-CTP fusion effectively increases stability without affecting secretion and activity. This study offers a sound strategy for improving the stability of neuritin protein and provides material conditions for further study of the function of neuritin.

Analysis of the in vitro function and internalization ability of a humanized EGFR antibody AE01 expressed by Chinese hamster ovary cells.

The primary objective of this study was to obtain humanized EGFR antibody and to study it in vitro binding and endocytosis to A431 epidermoid carcinoma cells overexpressing EGFR. Firstly, humanized anti-EGFR AE01 was stably expressed in CHO system. The expression of AE01 was detected by SDS-PAGE and Western blot. The binding and endocytosis of AE01 were detected by flow cytometry and immunofluorescence assay. The results showed that: (1) Pure humanized AE01 was prepared, (2) AE01 specifically binds to A431 cells on the cell surface (EGFR-positive), but not binds to NIH 3T3 cells (EGFR-negative), (3) AE01 can effectively inhibit the proliferation of A431 cells, and (4) AE01 binds to A431 cell surface triggered internalization. The antibody is expected to be a candidate molecule for EGFR overexpressed cancer cell targeted therapeutic vectors.

Protein overproduction alters exosome secretion in Chinese hamster ovary cells.

Despite the abundance of available cell lines, nearly 70% of all recombinant therapeutic proteins today are produced in Chinese hamster ovary (CHO) cells. The impact of protein overproduction on the secretion of exosomes by CHO cells has been investigated here. Increased secretion of extracellular vesicles (EVs) by protein overexpressing CHO cells was demonstrated with protein content assay, nanoparticle tracking analysis, and capillary electrophoresis. Our results revealed that a protein overproduction might induce EVs secretion, which might be accompanied by the sequestration and loading of overexpressed proteins into the exosomes. These findings are of vital importance for the manufacturing of therapeutics in CHO expression systems due to the risk of product loss during downstream processing of culture medium as well as the application of exosomes as nanocarriers of therapeutic proteins. The study indicates also the importance of culturing process control.