Gyun Min Lee

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

Chinese hamster ovary (CHO) cells are the preferred cell factory for the production of therapeutic glycoproteins. Although efforts primarily within bioprocess optimization have led to increased product titers of recombinant proteins (r-proteins) expressed in CHO cells, post-transcriptional bottlenecks in the biosynthetic pathway of r-proteins remain to be solved. To this end, the ectopic expression of transgenes (effector genes) offers great engineering potential. However, studies on effector genes have in some cases led to inconsistent results. Whereas this can in part be attributed to product specificity, other experimental and cellular factors are likely important contributors to these conflicting results. Here, these factors are reviewed and discussed with the objective of guiding future studies on effector genes.

Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment.

Targeted gene integration into site-specific loci can be achieved in Chinese hamster ovary (CHO) cells via CRISPR/Cas9 genome editing technology and the homology-directed repair (HDR) pathway. The low efficiency of HDR often requires antibiotic selection, which limits targeted integration of multiple genes at multiple sites. To improve HDR-mediated targeted integration, while avoiding the use of selection markers, chemical treatment for increased HDR, and fluorescent enrichment of genome-edited cells was assessed in CHO cells. Chemical treatment did not improve HDR-mediated targeted integration. In contrast, fluorescent markers in Cas9 and donor constructs enable FACS enrichment, resulting in a threefold increase in the number of cells with HDR-mediated genome editing. Combined with this enrichment method, large transgenes encoding model proteins (including an antibody) were successfully targeted integrated. This approach provides a simple and fast strategy for targeted generation of stable CHO production cell lines in a rational way. Biotechnol. Bioeng. 2016;113: 2518-2523.

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality.

Baicalein Reduces Oxidative Stress in CHO Cell Cultures and Improves Recombinant Antibody Productivity

Oxidative stress that naturally accumulates in the endoplasmic reticulum (ER) as a result of mitochondrial energy metabolism and protein synthesis can disturb the ER function. Because ER have a responsibility on the protein synthesis and quality control of the secreted proteins, ER homeostasis has to be well maintained. When H2 O2 , an oxidative stress inducer, is added to recombinant Chinese hamster ovary (rCHO) cell cultures, it reduced cell growth, monoclonal antibody (mAb) production, and galactosylated form of mAb in a dose-dependent manner. To find an effective antioxidant for rCHO cell cultures, six antioxidants (hydroxyanisole, N-acetylcysteine, baicalein, berberine chloride, kaempferol, and apigenin) with various concentrations are examined individually as chemical additives to rCHO cell cultures producing mAb. Among these antioxidants, baicalein shows the best mAb production performance. Addition of baicalein significantly reduced the expression level of BiP and CHOP along with reduced reactive oxygen species level, suggesting oxidative stress accumulated in the cells can be relieved using baicalein. As a result, addition of baicalein in batch cultures resulted in 1.7-1.8-fold increase in the maximum mAb concentration (MMC), while maintaining the galactosylation of mAb. Likewise, addition of baicalein in fed-batch culture resulted in 1.6-fold increase in the MMC while maintaining the galactosylation of mAb. Taken together, the results obtained here demonstrate that baicalein is an effective antioxidant to increase mAb production in rCHO cells.

Anti-Apoptosis Engineering for Improved Protein Production from CHO Cells

Improving the time integral of viable cell concentration by overcoming cell death, namely apoptosis, is one of the widely used strategies for efficient production of therapeutic proteins. By establishing stable cell lines that overexpress anti-apoptotic genes or down-regulate pro-apoptotic genes, the final product yields can be enhanced as cells become more resistance to environmental stresses. From the selection of high-expressing clones to verification of anti-apoptotic activity, the method to construct a stable anti-apoptotic cell line is discussed in this chapter.

Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile†

To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc‐fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc‐fusion protein productivity (qFc) but a decreased proportion of acidic isoforms and sialic acid content of the Fc‐fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc‐fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N‐glycosylation‐related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom‐designed color‐coded probes. After 3 days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5‐fold of the control, and all these genes were down‐regulated. N‐linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di‐, tri‐, tetra‐) and tetra‐antennary N‐linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra‐antennary N‐linked glycans (P ≤ 0.05), while it increased the expression of the N‐glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N‐glycan biosynthesis pathway correlated with reduced sialic acid content of Fc‐fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N‐glycosylation, especially sialylation, in rCHO cells. Biotechnol. Bioeng. 2017;114: 1721–1732.

Proteomic analysis of host cell protein dynamics in the supernatant of Fc-fusion protein-producing CHO DG44 and DUKX-B11 cell lines in batch and fed-batch cultures

Chinese hamster ovary (CHO) cells are the most widely used host cell lines for the commercial production of therapeutic proteins including Fc‐fusion proteins. During the culture of recombinant CHO (rCHO) cells, host cell proteins (HCPs), secreted from viable cells and released from dead cells, accumulate extracellularly, potentially impairing product quality. In this study, the HCPs that accumulated extracellularly in batch and fed‐batch cultures of Fc‐fusion protein‐producing rCHO cell lines (DG‐Fc and DUKX‐Fc) were identified and quantified using nanoflow liquid chromatography–tandem mass spectrometry (LC–MS/MS), followed by gene ontology and functional analysis. When the proteome database of Cricetulus griseus was used as a reference to identify the HCPs, more HCPs were identified for DG‐Fc (1632 HCPs in batch culture and 1733 HCPs in fed‐batch culture) than for DUKX‐Fc (1114 HCPs in batch culture and 1002 HCPs in fed‐batch culture). Clustering analysis of HCPs, which were classified into four clusters according to their concentration profiles during culture, showed that the concentration profiles of HCPs affecting the quality of Fc‐fusion proteins correlated with changes in Fc‐fusion protein quality. Taken together, the dataset of HCPs obtained in this study using the two different rCHO cell lines provides insights into the determination of appropriate target proteins to be removed during the culture and purification steps so as to ensure good Fc‐fusion protein quality. Biotechnol. Bioeng. 2017;114: 2267–2278.

Case study on human α1-antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme‐linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially available ELISA kits. We observed that estimations of recombinant human ø1‐antitrypsin (rø1AT) titer by Coomassie‐stained SDS‐PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit. This prompted us to develop two independent quantification assays based on biolayer interferometry and reversed‐phase high‐performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off‐the‐shelf ELISA kits and found that the ELISA kits led to inconsistent results. The data presented here show that recombinant protein titers determined by ELISA kits cannot be trusted per se. Consequently, any ELISA kit to be used for determining recombinant protein titer must be validated by a different, preferably orthogonal method.

Using Titer and Titer Normalized to Confluence Are Complementary Strategies for Obtaining Chinese Hamster Ovary Cell Lines with High Volumetric Productivity of Etanercept

The selection of clonally derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number are being used to assess suspension-adapted clones when grown statically in microtiter plates. However, no reported study so far has performed a direct head-to-head comparison of these two early reporters for predicting clone performance. Therefore, a screening platform for high-throughput analysis of titer and confluence of etanercept-producing clones is developed. Then an unbiased comparison of clone ranking based on either titer or titer normalized to confluence (TTC) is performed. Using two different suspension cultivation vessels, the authors demonstrate that titer- or TTC-based ranking gives rise to the selection of clones with similar volumetric productivity in batch cultures. Therefore, using both titer- and TTC-based ranking is proposed, allowing for selection of distinct clones with both high integral of viable cell density (IVCD) and high specific productivity features, respectively. This contributes to selection of a versatile panel of clones that can be further characterized and from which the final producer clone can be selected that best fits the production requirements.