Omics Approaches To Mammalian Cell Metabolic Engineering

https://digitalrepository.unm.edu/abq_mj_news/1035/thumbnail.jp

Tailoring antibody glycosylation via integrating genome and protein engineering to generate preferred glycoforms on the Fc region

One critical quality attribute of therapeutic antibodies is the glycosylation pattern at the Fc region.We combined genome editing of CHO cells and protein engineering of the IgG Fc region to allow antibodies presenting high level of galactosylation or exclusively α-2,6 sialylation. To generate IgG with high α-2,6 sialylation, we combined amino acid mutations in the Fc region of IgG and introduction of α-2,6 sialyltransferase in CHO to produce IgGs with significant levels of both α-2,6 and α-2,3 sialylation. Furthermore, to produce exclusively α-2,6 sialylation IgG in CHO, CRISPR/Cas9 was implemented to disrupt two dominant α-2,3 sialyltransferase genes (ST3GAL4 and ST3GAL6), then α-2,6 sialyltransferewas introduced in a α-2,3 sialylation knockout cell line. Notably, no α-2,3 linked sialic acids of IgG produced from the α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase overexpression pools were detected by HPLC sialic acid quantification after the α-2,3 linkage specific sialidase cleavage. Finally, glycosylation analysis of IgG with four amino acid mutations generated by an α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase overexpression stable CHO clone rendered \u3e75% of sialylated glycans, among which 62.5 % was biantennary disialylated glycans. Interestingly, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from CHO cells in conjunction with protein engineering of the Fc region produced IgGs with a great majority of bigalactosylated and fucosylated (G2F) glycoforms. Expression of the IgG with engineered Fc region (F241A) in triple gene knockout (FuT8-/-, ST3GAL4-/- and ST3GAL6-/-) CHO cells lowered the galactosylation content to 65% bigalactosylated glycoform (G2). However, overexpression of IgGs with four amino acid substitutions from the α-2,3 sialyltransferases knocked out CHO cells reconstituted the fraction of G2 glycoform back up to approximately 80%. Collectively, this study, to our knowledge, is the first attempt for generating highly galactosylated or solely α-2,6 sialylated N-glycans on antibodies in vivo, allowing researchers in both academia and industry to evaluate the significance of tailoring glycosylation on IgGs in biomedicine and biotechnology applications. References: Chung CY, Wang Q, Yang S, Ponce SA, Kirsch BJ, Zhang H, Betenbaugh MJ. Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells. Biotechnol Bioeng. 2017 Jul 7 Chung CY, Wang Q, Yang S, Yin B, Zhang H, Betenbaugh M. Integrated Genome and Protein Editing Swaps α-2,6 Sialylation for α-2,3 Sialic Acid on Recombinant Antibodies from CHO. Biotechnol J. 2017 Feb;12(2

Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog

<p>Abstract</p> <p>Background</p> <p>Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.</p> <p>Results</p> <p>Two genes, cyclin-dependent kinase like 3 (<it>cdkl3</it>) and cytochrome c oxidase subunit (<it>cox15</it>), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of <it>cdkl3 </it>had a greater impact than that of <it>cox15</it>. Moreover, flow cytometric analysis indicated that cells with an insert of <it>cdkl3 </it>were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of <it>cox15 </it>was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than <it>cdkl3</it>. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either <it>cdkl3 </it>or <it>cox15</it>.</p> <p>Conclusion</p> <p>These results are consistent with previous studies on the functionalities of <it>cdkl3 </it>and <it>cox15</it>. For instance, the effect of <it>cdkl3 </it>on cell growth is consistent with its homology to the <it>cdk3 </it>gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to <it>cox15 </it>expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.</p

Lipidomic analysis to enhance the understanding of Chinese Hamster ovary cells

Chinese Hamster Ovary (CHO) cell lines are common hosts for the production of biotherapeutic proteins. Achieving high level of specific protein production by CHO cell lines remains a challenge. In order to address this issue, we are incorporating lipidomic analyses to study the role of lipids played in CHO-S cells. In our study, we have applied chromatography (TLC) methods for lipid analysis in terms of lipid polarity. For polar lipids, 2-D HPTLC (2-dimensional high performance TLC) was used instead of conventional 1D- TLC by virtue of its high separation capacity. The eluting solvent system was optimized for the 1st and 2nd dimension, respectively. Neutral lipids were separated on 1-D HPTLC with the optimal elution solvent of hexane-diethyl ether-acetic acid. The lipid spots on the TLC plates were stained by 0.2% of 2,7-dichlorofluorescein dissolved in ethanol solution and illuminated with UV. Multiple lipid standards were also run to correctly identify the lipid spots and the fluorescence of lipid spots was semi-quantitatively measured with ImageJ. By optimization of TLC conditions, the lipids of CHO-S cell line were separated successfully and the lipid contents were semi-quantified. From neutral lipids result, we observed high level of certain lipids in CHO-S cell lines. We will further investigate which lipid play a key role in various cell processes

Improving production of retroviral vector from Pg13 cells for T cell therapy

Adoptive T-Cell therapy is a growing field for cancer treatment using the patient’s immune system to battle the cancer cells. Tumor specific T cells are either isolated from a tumor or created by modifying the T cells and after expansion are administered to the patient. The modifications include adding specific T cell receptors (TCR) or chimeric antigen receptors (CAR) by way of retroviral vector, lentiviral vector, or other method. One method is to use PG13 cells, which are derivatives of NIH3T3 mouse fibroblasts, to stably produce a retroviral vector that is used to transduce the T cell. PG13 cells are anchorage dependent cells that grow in roller bottles or cell factories to produce the viral vector and recently in a fixed bed bioreactor. To improve the production of the viral vector we explore the possibility of its production using PG13 cells grown on microcarriers in a bioreactor. Microcarriers are small, approximately 100-300 µm, charged beads that support the attachment of the cells and are suspended in the growth media in the bioreactor that provide controlled growth conditions. In this way parameters, such as oxygen concentration, pH, and nutrient are monitored and controlled. The result is higher cell concentration and consequently virus titer. There was no effect on the specific virus titer or the efficacy of the vector in transducing t cells indicating that using microcarriers in a bioreactor is a good method for scaling up stable production of gamma retroviral vector in PG13 cells

Intact glycopeptide analysis of recombinant protein from CHO cells

The quality of recombinant glycoproteins including antibodies and other biologics is dictated by their glycan profiles. What is missing is how to analyze these glycans rapidly for process improvement and control applications. Conventional glycan analysis involves the release of glycans, which rarely captures the glycan site-specific information. Intact glycopeptide analysis in which glycans are retained on the peptide provides insights into the glycan structure and the glycosylation site information simultaneously. This information can reveal additional details about site occupancy and cellular glycosylation of proteins. Avoiding glycan release and some modifications and labeling steps in our intact glycopeptide analysis can result in a shorter sample preparation time than conventional glycan analysis methods. Compared to peptide mapping using LC-MS to decipher protein amino acid sequence in proteomics, this analysis focuses on glycopeptide profiling following protease-digestion. With the aid of LC-MS/MS, we are able to obtain targeted glycoprotein sequence information, glycan profiles and glycan distribution at specific sites. Here we present the application of glycopeptide analysis for model AMBIC and other proteins from CHO-GS and CHO-K1 cells. The site-specific glycosylation patterns of our model proteins EPO-Fc and EPO are characterized. Further, we examine the impact of media formulation and additives on the glycan profiles for these proteins. Please click Additional Files below to see the full abstract

Identifying Hipk1 as a target of Mir-22-3p enhancing recombinant protein production from Hek 293 by using microarray and Htp sirna screen

Enhancing protein production in mammalian cells is of interest in the biomedical field for a variety of reasons, including structural studies and antibody production. Using small non-protein coding RNA such as microRNA has recently been a promising method of increasing protein expression. A high throughput human microRNA screen in HEK 293 cells previously identified miRNA 22-3p as a promising candidate for increasing recombinant protein expression. This microRNA enhanced the expression of luciferase, two hard-to-express membrane proteins and a secreted hFc-fusion protein. In order to explore the mechanisms of this increase in protein production and to understand the intracellular events, we conducted a gene expression analysis of cells transfected with a mir-22-3p mimic against a negative control. Following the microarray analysis, several genes that were differentially regulated were identified. These were cross-referenced with predicted mir-22-3p targets along with the results of a high throughput siRNA screen. We will present our selected gene, HIPK1, and its possible involvement in the process of enhanced cells productivity

Model-based analysis of N-glycosylation in Chinese hamster ovary cells

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower likelihood of causing immunogenic responses. Because glycan structures are the product of the concerted action of intracellular enzymes, it is difficult to predict a priori how the effects of genetic manipulations alter glycan structures of cells and therapeutic properties. For that reason, quantitative models able to predict glycosylation have emerged as promising tools to deal with the complexity of glycosylation processing. For example, an earlier version of the same model used in this study was used by others to successfully predict changes in enzyme activities that could produce a desired change in glycan structure. In this study we utilize an updated version of this model to provide a comprehensive analysis of N-glycosylation in ten Chinese hamster ovary (CHO) cell lines that include a wild type parent and nine mutants of CHO, through interpretation of previously published mass spectrometry data. The updated N-glycosylation mathematical model contains up to 50,605 glycan structures. Adjusting the enzyme activities in this model to match N-glycan mass spectra produces detailed predictions of the glycosylation process, enzyme activity profiles and complete glycosylation profiles of each of the cell lines. These profiles are consistent with biochemical and genetic data reported previously. The model-based results also predict glycosylation features of the cell lines not previously published, indicating more complex changes in glycosylation enzyme activities than just those resulting directly from gene mutations. The model predicts that the CHO cell lines possess regulatory mechanisms that allow them to adjust glycosylation enzyme activities to mitigate side effects of the primary loss or gain of glycosylation function known to exist in these mutant cell lines. Quantitative models of CHO cell glycosylation have the potential for predicting how glycoengineering manipulations might affect glycoform distributions to improve the therapeutic performance of glycoprotein products