Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase-mediated cassette exchange system.

Cell line development (CLD) by random integration (RI) can be labor intensive, inconsistent, and unpredictable due to uncontrolled gene integration after transfection. Unlike RI, targeted integration (TI) based CLD introduces the antibody-expressing cassette to a predetermined site by recombinase-mediated cassette exchange (RMCE). The key to success for the development of a TI host for therapeutic antibody production is to identify a transcriptionally active hotspot that enables highly efficient RMCE and antibody expression with good stability. In this study, a genome wide search for hotspots in the Chinese hamster ovary (CHO)-K1-M genome by either RI or PiggyBac (PB) transposase-based integration has been described. Two CHO-K1-M derived TI host cells were established with the Cre/Lox RMCE system and are described here. Both TI hosts contain a GFP-expressing landing pad flanked by two incompatible LoxP recombination sites (L3 and 2L). In addition, a third incompatible LoxP site (LoxFAS) is inserted in the GFP landing pad to enable an innovative two-plasmid based RMCE strategy, in which two separate vectors can be targeted to a single locus simultaneously. Cell lines generated by the TI system exhibit comparable or higher productivity, better stability and fewer sequence variant (SV) occurrences than the RI cell lines.

Development and characterization of an automated imaging workflow to generate clonally-derived cell lines for therapeutic proteins.

In the development of biopharmaceutical products, the expectation of regulatory agencies is that the recombinant proteins are produced from a cell line derived from a single progenitor cell. A single limiting dilution step followed by direct imaging, as supplemental information, provides direct evidence that a cell line originated from a single progenitor cell. To obtain this evidence, a high-throughput automated imaging system was developed and characterized to consistently ensure that cell lines used for therapeutic protein production are clonally-derived. Fluorescent cell mixing studies determined that the automated imaging workflow and analysis provide ∼95% confidence in accurately and precisely identifying one cell in a well. Manual inspection of the images increases the confidence that the cell line was derived from a single-cell to >99.9%. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:584-592, 2018.

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies.

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies-mAb1, mAb2, and mAb3-at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449-1455, 2017.

Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

Industry view on the relative importance of "clonality" of biopharmaceutical-producing cell lines.

Recently, several health authorities have requested substantial detail from sponsor firms regarding the practices employed to generate the production cell line for recombinant DNA-(rDNA) derived biopharmaceuticals. Two possible inferences from these regulatory agency questions are that (1) assurance of "clonality" of the production cell line is of major importance to assessing the safety and efficacy of the product and (2), without adequate proof of "clonality", additional studies of the cell line and product are often required to further ensure the product's purity and homogeneity. Here we address the topic of "clonality" in the broader context of product quality assurance by current technologies and practices, as well as discuss some of the relevant science and historical perspective. We agree that the clonal derivation of a production cell line is one factor with potential impact, but it is only one of many factors. Further, we believe that regulatory emphasis should be primarily placed on ensuring product quality of the material actually administered to patients, and on ensuring process consistency and implementing appropriate control strategies through the life cycle of the products.

Effects of copper on CHO cells: cellular requirements and product quality considerations.

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes.

Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3.

Fast identification of reliable hosts for targeted cell line development from a limited-genome screening using combined φC31 integrase and CRE-Lox technologies.

The use of targeted integration (TI) in cell line development (CLD) usually introduces one copy of a recombinant gene into a predetermined transcriptionally active locus. This reduces the heterogeneity typically associated with traditional random integration (RI) CLD with regards to varied productivity and instability, resulting from diverse chromosomal influences, varied copy numbers, and repeat-induced rearrangement. As such, TI CLD offers the hope of a predictable and consistent CLD process for establishing stable clones. However, given the low copy number, cell lines established from a TI CLD process tend to exhibit low productivity. Here, we describe our nonviral-based approach for quickly establishing and identifying TI hosts from a limited genome screening. Importantly, the TI hosts identified are consistent and reliable in supporting the production of diverse antibodies regardless of antibody subclass (IgG1 vs. IgG4) or prior traditional CLD performance (relatively easy vs. difficult to express antibodies). Moreover, an approximately twofold increase in titer can be achieved by using a CRE recombinase-mediated cassette exchange (RMCE) strategy with an exchange vector carrying two units of the antibody gene. Two RMCE hosts that were established were able to produce up to ∼ 1.7 and 2 g/L of antibodies in nonoptimized fed-batch shake flask production cultures with chemically defined media. Potentially, this strategy may be applied to the production of bispecific antibodies with a fast turnaround time.

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell.

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass.

Controlling glycation of recombinant antibody in fed-batch cell cultures.

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases.

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells.

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6-thioguanine (6-TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6-TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb-A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb-B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity.

Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical.

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.

High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain.

During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed E. coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in degP strains. However, light chain proteolysis was substantially decreased in prc and prc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E. coli, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations.

The cryptic ushA gene (ushA(c)) in natural isolates of Salmonella enterica (serotype Typhimurium) has been inactivated by a single missense mutation.

Two mutational mechanisms, both supported by experimental studies, have been proposed for the evolution of new or improved enzyme specificities in bacteria. One mechanism involves point mutation(s) in a gene conferring novel substrate specificity with partial or complete loss of the original (wild-type) activity of the encoded product. The second mechanism involves gene duplication followed by silencing (inactivation) of one of these duplicates. Some of these 'silent genes' may still be transcribed and translated but produce greatly reduced levels of functional protein; gene silencing, in this context, is distinct from the more common associations with bacterial partitioning sequences, and with genes which are no longer transcribed or translated. Whereas most Salmonella enterica strains are ushA(+), encoding an active 5'-nucleotidase (UDP-sugar hydrolase), some natural isolates, including most genetically related strains of serotype Typhimurium, have an ushA allele (designated ushA(c)) which produces a protein with, comparatively, very low 5'-nucleotidase activity. Previous sequence analysis of cloned ushA(c) and ushA(+) genes from serotype Typhimurium strain LT2 and Escherichia coli, respectively, did not reveal any changes which might account for the significantly different 5'-nucleotidase activities. The mechanism responsible for this reduced activity of UshA(c) has hitherto not been known. Sequence analysis of Salmonella ushA(+) and ushA(c) alleles indicated that the relative inactivity of UshA(c) may be due to one, or more, of four amino acid substitutions. One of these changes (S139Y) is in a sequence motif that is conserved in 5'-nucleotidases across a range of diverse prokaryotic and eukaryotic species. Site-directed mutagenesis confirmed that a Tyr substitution of Ser-139 in Salmonella UshA(+) was solely responsible for loss of 5'-nucleotidase activity. It is concluded that the corresponding single missense mutation is the cause of the UshA(c) phenotype. This is the first reported instance of gene inactivation in natural isolates of bacteria via a missense mutation. These results support a model of evolution of new enzymes involving a 'silent gene' which produces an inactive, or relatively inactive, product, and are also consistent with the evolution of a novel, but unknown, enzyme specificity by a single amino acid change.