Nature as blueprint: Global phenotype engineering of CHO production cells based on a multi-omics comparison with plasma cells.

Especially for the production of artificial, difficult to express molecules a further development of the CHO production cell line is required to keep pace with the continuously increasing demands. However, the identification of novel targets for cell line engineering to improve CHO cells is a time and cost intensive process. Since plasma cells are evolutionary optimized for a high antibody expression in mammals, we performed a comprehensive multi-omics comparison between CHO and plasma cells to exploit optimized cellular production traits. Comparing the transcriptome, proteome, miRNome, surfaceome and secretome of both cell lines identified key differences including 392 potential overexpression targets for CHO cell engineering categorized in 15 functional classes like transcription factors, protein processing or secretory pathway. In addition, 3 protein classes including 209 potential knock-down/out targets for CHO engineering were determined likely to affect aggregation or proteolysis. For production phenotype engineering, several of these novel targets were successfully applied to transient and transposase mediated overexpression or knock-down strategies to efficiently improve productivity of CHO cells. Thus, substantial improvement of CHO productivity was achieved by taking nature as a blueprint for cell line engineering.

Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing.

Genetically modified CHO cell lines are traditionally used for the production of biopharmaceuticals. However, an in-depth molecular understanding of the mechanism and exact position of transgene integration into the genome of pharmaceutical manufacturing cell lines is still scarce. Next-generation sequencing (NGS) holds great promise for strongly facilitating the understanding of CHO cell factories, as it has matured to a powerful and affordable technology for cellular genotype analysis. Targeted Locus Amplification (TLA) combined with NGS allows for robust detection of genomic positions of transgene integration and structural genomic changes occurring upon stable integration of expression vectors. TLA was applied to generate comparative genomic fingerprints of several CHO production cell lines expressing different monoclonal antibodies. Moreover, high producers resulting from an additional round of transfection of an existing cell line (supertransfection) were analyzed to investigate the integrity and the number of integration sites. Our analyses enabled detailed genetic characterization of the integration regions with respect to the number of integrates and structural changes of the host cell's genome. Single integration sites per clone with concatenated transgene copies could be detected and were in some cases found to be associated with genomic rearrangements, deletions or translocations. Supertransfection resulted in an increase in titer associated with an additional integration site per clone. Based on the TLA fingerprints, CHO cell lines originating from the same mother clone could clearly be distinguished. Interestingly, two CHO cell lines originating from the same mother clone were shown to differ genetically and phenotypically despite their identical TLA fingerprints. Taken together, TLA provides an accurate genetic characterization with respect to transgene integration sites compared with conventional methods and represents a valuable tool for a comprehensive evaluation of CHO production clones early in cell line development.

Loss of a newly discovered microRNA in Chinese hamster ovary cells leads to upregulation of N-glycolylneuraminic acid sialylation on monoclonal antibodies.

Chinese hamster ovary (CHO) cells are known not to express appreciable levels of the sialic acid residue N-glycolylneuraminic acid (NGNA) on monoclonal antibodies. However, we actually have identified a recombinant CHO cell line expressing an IgG with unusually high levels of NGNA sialylation (>30%). Comprehensive multi-OMICs based experimental analyses unraveled the root cause of this atypical sialylation: (1) expression of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene was spontaneously switched on, (2) CMAH mRNA showed an anti-correlated expression to the newly discovered Cricetulus griseus (cgr) specific microRNA cgr-miR-111 and exhibits two putative miR-111 binding sites, (3) miR-111 expression depends on the transcription of its host gene SDK1, and (4) a single point mutation within the promoter region of the sidekick cell adhesion molecule 1 (SDK1) gene generated a binding site for the transcriptional repressor histone H4 transcription factor HINF-P. The resulting transcriptional repression of SDK1 led to a downregulation of its co-expressed miR-111 and hence to a spontaneous upregulation of CMAH expression finally increasing NGNA protein sialylation.

Deciphering integration loci of CHO manufacturing cell lines using long read nanopore sequencing.

Despite advances in genetic characterization of Chinese hamster ovary (CHO) cell lines regarding identification of integration sites using next generation sequencing, e.g. targeted locus amplification sequencing (TLA-seq), the concatemer structure of the integrated vectors remains elusive. Here, the entire integration locus of two CHO manufacturing cell lines was reconstructed combining CRISPR/Cas9 target enrichment, nanopore sequencing and the Canu de novo assembly pipeline. An IgG producing CHO cell line integrated 3 vector copies, which were near full-length and contained all relevant vector elements such as transgenes and their promoters on each of the vector copies. In contrast, a second CHO cell line producing a bivalent bispecific antibody integrated 7 highly fragmented vector copies in different orientations leading to head-to-head and tail-to-tail fusions. The size of the vector fragments ranged from 3.0 to 11.4 kbp each carrying 1-3 transgenes. The breakpoints of the genome-vector and vector-vector junctions were validated using Sanger sequencing and Southern blotting. A comparison to TLA-seq data confirmed the genomic breakpoints, but most of the breakpoints of the vector-vector fusions were missed by TLA-seq. For the first time, the complete transgene locus of CHO manufacturing cell lines could be deciphered. Strikingly, the application of the nanopore long-read sequencing technology led to novel insights into the complexity of genomic transgene integrations of CHO manufacturing cell lines generated via random integration.

High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells.

Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed-batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5 mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale-up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next-generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21-mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor-like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine-enriched cell culture media.

A blueprint from nature: miRNome comparison of plasma cells and CHO cells to optimize therapeutic antibody production.

Chinese Hamster Ovary (CHO) cells are the most frequently used biopharmaceutical production hosts, although industry is presently suffering from their variable recombinant product quality, insufficient long-term stability and low productivity. Here, we present an effort to address overall cell line engineering by a novel bottom-up microRNA (miRNA) screening approach. miRNAs are small non-coding RNAs known to regulate global gene expression at the post-transcriptional level and have proved to serve as promising tools for cell line engineering for over a decade. Here the miRNome of plasma cells (PCs) has been analyzed as the natural blueprint for optimized production and secretion of antibodies. Performing comparative miRNome cross-species expression analysis of four murine/human PC-derived (PCD) and two CHO cell lines showed 147 conserved miRNAs to be differentially expressed between PCDs and CHOs. Conducting a targeted miRNA screen of this PC-specific miRNA subset revealed 14 miRNAs to improve bioprocess relevant parameters in CHO cells, among them the PC-characteristic miR-183 cluster. Finally, miRNA target prediction tools and transcriptome analysis were combined to elucidate differentially regulated lysine degradation and fatty acid metabolism pathways in monoclonal antibody (mAb) expressing CHO-DG44 and CHO-K1 cells, respectively. Thus, substantial new insights into molecular and cellular mechanisms of biopharmaceutical production cell lines can be gained by targeted bottom-up miRNA screenings.