Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics.

A clonally derived (or "monoclonal") cell line is a cell population derived from a single progenitor cell. Clonally derived cell lines are required for many biotechnological applications. For instance, recombinant mammalian cells used to produce therapeutic proteins are expected by regulatory authorities to be clonally derived. Assurance of clonal derivation (or "clonality") is usually obtained from the characterization of the procedure used for cell cloning, for instance by assessing the success rate of single-cell sorting but not by assessing the cell line itself. We have developed a method to assess clonal derivation directly from the genetic makeup of cells. The genomic test of clonality is based on whole-genome sequencing and statistical analysis of single nucleotide variants. This approach quantifies the clonal fractions present in nonclonal samples and it provides a measure of the probability that a cell line is derived from a single cell. Upon experimental validation of the test, we show that it is highly accurate and that it can robustly detect minor clonal fractions of as little as 1% of the cell population. Moreover, we find that it is applicable to various cell line development protocols. This approach can simplify development protocols and shorten timelines while ensuring clonal derivation with high confidence.

Transient vitamin B5 starving improves mammalian cell homeostasis and protein production.

Maintaining a metabolic steady state is essential for an organism's fitness and survival when confronted with environmental stress, and metabolic imbalance can be reversed by exposing the organism to fasting. Here, we attempted to apply this physiological principle to mammalian cell cultures to improve cellular fitness and consequently their ability to express recombinant proteins. We showed that transient vitamin B5 deprivation, an essential cofactor of central cellular metabolism, can quickly and irreversibly affect mammalian cell growth and division. A selection method was designed that relies on mammalian cell dependence on vitamin B5 for energy production, using the co-expression of the B5 transporter SLC5A6 and a gene of interest. We demonstrated that vitamin B5 selection persistently activates peroxisome proliferator-activated receptors (PPAR), a family of transcription factors involved in energy homeostasis, thereby altering lipid metabolism, improving cell fitness and therapeutic protein production. Thus, stable PPAR activation may constitute a cellular memory of past deprivation state, providing increased resistance to further potential fasting events. In other words, our results imply that cultured cells, once exposed to metabolic starvation, may display an improved metabolic fitness as compared to non-exposed cells, allowing increased resistance to cellular stress.

Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells.

Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy-to-express antibody, whereas Erp27 and Erp57 co-overexpression increased cell density, viability, and the yield of difficult-to-express proteins. Foxa1 overexpression increased cell density, cell viability, and easy- and difficult-to-express protein yields, whereas it decreased reactive oxygen species late in fed-batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult- and/or easy-to-express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.

Influence of cytoskeleton organization on recombinant protein expression by CHO cells.

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase-activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein-expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by-products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.

Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies.

Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan "hole" naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.

Automated microfluidic sorting of mammalian cells labeled with magnetic microparticles for those that efficiently express and secrete a protein of interest.

We developed a method for the fast sorting and selection of mammalian cells expressing and secreting a protein at high levels. This procedure relies on cell capture using an automated microfluidic device handling antibody-coupled magnetic microparticles and on a timed release of the cells from the microparticles after capture. Using clinically compatible materials and procedures, we show that this approach is able to discriminate between cells that truly secrete high amounts of a protein from those that just display it at high levels on their surface without properly releasing it. When coupled to a cell colony imaging and picking device, this approach allowed the identification of CHO cell clones secreting a therapeutic protein at high levels that were not achievable without the cell sorting procedure. Biotechnol. Bioeng. 2017;114: 1791-1802. © 2017 Wiley Periodicals, Inc.

MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384-396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

MAR elements and transposons for improved transgene integration and expression.

Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy.

Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin-proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα-γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber et al. Proc Natl Acad Sci USA 104:4407-4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα-γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoproteins.

Protein N-glycosylation, protein folding, and protein quality control.

Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.

Protein quality control: the who's who, the where's and therapeutic escapes.

In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.

Competition between calnexin and BiP in the endoplasmic reticulum can lead to the folding or degradation of human thyroperoxidase.

Previous studies on the fate of human thyroperoxidase (hTPO) molecules have shown that, after being synthesized, these glycoproteins interact with calnexin and calreticulin and that only some of them are able to acquire a partially folded structure. The aim of the present study was to further investigate the potential role of BiP, another major protein chaperon. Co-immunoprecipitation experiments showed the occurrence of interactions between hTPO and BiP. Pulse-chase studies showed that, when hTPO was expressed in a Chinese hamster ovary cell line overexpressing 5 times more BiP than the parent cells, the rate of hTPO recognized by a monoclonal antibody directed against a conformational structure decreased by 50% after 5 h of chase. Overexpression of the BiP-ATPase mutant G37T also led to a decrease in the correct folding rate of hTPO. When this protein was pulsed in the presence of 35S-(Met + Cys) and the reducing agent dithiotreitol and then chased in a culture medium without dithiothreitol, a 2.5-fold decrease in the correct folding rate was observed in cells overexpressing BiP, whereas co-overexpression of calnexin and Erp57 led to an increase in both the unfolded and partially folded form of hTPO after the pulse step. All of these findings show that BiP and calnexin have opposite effects on the folding behavior of hTPO and that the action of specific molecular chaperones may therefore crucially determine the fate of glycoproteins.

Endoproteolytic cleavage of human thyroperoxidase: role of the propeptide in the protein folding process.

Human thyroperoxidase (hTPO), the key enzyme involved in thyroid hormone synthesis, is synthesized in the form of a 933-amino acid polypeptide that subsequently undergoes posttranslational modifications such as N- and O-glycosylation and heme fixation. In the present study, it was established that the N-terminal part of hTPO is cleaved during the maturation of the enzyme. In the first set of experiments performed in this study, Chines hamster ovary (CHO) cells transfected with hTPO cDNA generated four different species after deglycosylation, namely a 98-kDa species, which corresponds to the full-length deglycosylated hTPO, and two 94-kDa and one 92-kDa species, which were truncated in the N-terminal parts. The three latter forms were detected only at the cell surface. A proprotein convertase inhibitor prevented these cleavages, and experiments using monensin and brefeldin A showed that they occurred in a post-endoplasmic reticulum compartment. Site-directed mutagenesis studies were performed in which Arg65 was identified as one of the cleavage sites. In the second part of the study, hTPO from human thyroid glands was purified using a monoclonal antibody recognizing the folded form of hTPO. Amino acid determination showed that the N-terminal part of this protein begins at Thr109. This cleavage process differs from that observed in CHO cells. The fact that this hTPO was endoglucosaminidase H-sensitive indicated that the cleavage of the propeptide occurs in the endoplasmic reticulum. To analyze the role of the hTPO prosequence, cDNAs with and without prosequence (Cys15-Lys108) were transfected into CHO cells. hTPO propeptide deletion drastically decreased the proportion of the folded hTPO form, and under these conditions the cell surface activity disappeared completely. These results strongly suggest that the prosequence plays a crucial role as an intramolecular chaperone, facilitating the folding of hTPO.

Differential expression of thyroperoxidase mRNA splice variants in human thyroid tumors.

The levels of human thyroperoxidase (hTPO) mRNA expression and the rates of hTPO mRNA with alternatively spliced exons 10, 14, and 16 were analyzed in normal, benign, and malignant thyroid tissues (13 normal thyroid tissues, 9 adenomas, 4 papillary carcinomas, 11 follicular variant of papillary carcinomas, 16 minimally invasive follicular carcinomas, 6 widely invasive follicular carcinomas) using a semi-quantitative reverse-transcription polymerase chain reaction procedure. The level of hTPO mRNA decreased in the follicular variant of papillary carcinomas and in minimally invasive follicular carcinomas and was more heterogeneous in the other pathological tissues than in normal tissues. Based on the mean values recorded, the splicing of exons 10 and 16 increased by at least 50% in all the carcinomas, as well as in the benign tissues in the case of exon 10. By contrast, no significant increase was observed in the splicing of exon 14 except in the case of the follicular variant of papillary carcinomas. In conclusion, the results of this study show that the splicing of hTPO increases in benign and malignant thyroid tissues. This event might partly explain the decrease in both the quantity and the level of activity of hTPO observed in thyroid cancer due to the loss of stability of the spliced isoforms. In addition, an increase in the alternative splicing of other mRNAs may contribute to the process of malignancy.

Increasing diversity of human thyroperoxidase generated by alternative splicing. Characterized by molecular cloning of new transcripts with single- and multispliced mRNAs.

The human thyroperoxidase (hTPO) gene is composed of 17 exons. The longest complete cDNA sequence determined so far contains a full-length hTPO (TPO1) encoding a 933-amino acid polypeptide. Several mRNA species encoding for hTPO isoforms are present in normal thyroid tissues, including TPO2 with exon 10 deleted and TPOzanelli with exon 16 deleted. In the present study, we established the existence of two new single-spliced transcripts, TPO4 and TPO5, lacking exons 14 and 8, respectively. Upon transfecting the TPO4 cDNA into Chinese hamster ovary cells, it was observed that TPO4 is able to reach the cell surface, is enzymatically active, and is able to be recognized by a panel of 12 monoclonal antibodies directed against hTPO, whereas TPO5 does not fold correctly and is unable to reach the cell surface. In normal tissues, the expression of TPO4 mRNA was examined by performing quantitative reverse transcription PCR. This deleted TPO mRNA amounted to 32 +/- 11% of the total TPO mRNAs. In the same tissues, the TPO2, TPOzanelli, and TPO5 amounted to 35 +/- 12%, 36 +/- 14%, and approximately 10%, respectively. The sum of these four species (not including TPO1) was more than 100%, possibly due to the presence of multispliced mRNAs. This possibility was tested, and three new variants were identified: TPO2/3, lacking exons 10 and 16, TPO2/4, lacking exons 10 and 14, and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16. In conclusion, these results indicate the existence of five new transcripts. One of them, TPO4, codes for an enzymatically active protein, whereas TPO5 is unable to fold correctly. The functional significance of the other newly spliced mRNA variants still remains to be elucidated, but these results might help to explain the heterogeneity of the hTPO purified from the thyroid gland.