Identification of Mispairing Omic Signatures in Chinese Hamster Ovary (CHO) Cells Producing a Tri-Specific Antibody.

Monoclonal antibody-based therapy has shown efficacy against cancer, autoimmune, infectious, and inflammatory diseases. Multispecific antibodies (MsAbs), including trispecifics (tsAbs), offer enhanced therapeutic potential by targeting different epitopes. However, when co-expressed from three or more different polypeptide chains, MsAb production can lead to incorrect chain assembly and co-production of mispaired species with impaired biological activity. Moreover, mispairing carries significant challenges for downstream purification, decreasing yields and increasing the cost of bioprocess development. In this study, quantitative transcriptomics and proteomics analyses were employed to investigate which signaling pathways correlated with low and high mispairing clone signatures. Gene and protein expression profiles of Chinese hamster ovary (CHO) clones producing an tsAb were analyzed in the exponential growth and stationary (tsAb production) phase of fed-batch culture. Functional analysis revealed activated endoplasmic reticulum stress in high mispairing clones in both culture phases, while low mispairing clones exhibited expression profiles indicative of activated protein translation, as well as higher endocytosis and target protein degradation, suggesting the clearance of unfolded proteins through ubiquitin-mediated mechanisms. In addition, through transcriptomic profiling, we identified a group of genes that have the potential to be used as a biomarker panel tool for identifying high mispairing levels in the early stages of bioprocess development.

LncRNA analysis of mAb producing CHO clones reveals marker and engineering potential.

Long non-coding RNAs (lncRNAs) are a potential new cell line engineering tool for improvement of yield and stability of CHO cells. In this study, we performed RNA sequencing of mAb producer CHO clones to study the lncRNA and protein coding transcriptome in relation to productivity. First, a robust linear model was used to identify genes correlating to productivity. To unravel specific patterns in expression of these genes, we employed weighted gene coexpression analysis (WGCNA) to find coexpressed modules, looking both for lncRNAs and coding genes. There was little overlap in the genes associated with productivity between the two products studied, possibly due to the difference in absolute range of productivity between the two mAbs. Therefore, we focused on the product with higher productivity and stronger candidate lncRNAs. To evaluate their potential as engineering targets, these candidate lncRNAs were transiently overexpressed or deleted by stable CRISPR Cas9 knock out both in a high and a low productivity subclone. We found that the thus achieved expression level of the identified lncRNAs, as confirmed by qPCR, does correlate well to productivity, so that they represent good markers that may be used for early clone selection. Additionally, we found that the deletion of one tested lncRNA region decreased viable cell density (VCD), prolonged culture time and increased cell size, final titer and specific productivity per cell. These results demonstrate the feasibility and usefulness of engineering lncRNA expression in production cell lines.

Implementation of Plate Imaging for Demonstration of Monoclonality in Biologics Manufacturing Development.

Monoclonality of mammalian cell lines used for production of biologics is a regulatory expectation and one of the attributes assessed as part of a larger process to ensure consistent quality of the biologic. Historically, monoclonality has been demonstrated through statistics generated from limiting dilution cloning or through verified flow cytometry methods. A variety of new technologies are now on the market with the potential to offer more efficient and robust approaches to generating and documenting a clonal cell line.Here we present an industry perspective on approaches for the application of imaging and integration of that information into a regulatory submission to support a monoclonality claim. These approaches represent the views of a consortium of companies within the BioPhorum Development Group and include case studies utilising imaging technology that apply scientifically sound approaches and efforts in demonstrating monoclonality. By highlighting both the utility of these alternative approaches and the advantages they bring over the traditional methods, as well as their adoption by industry leaders, we hope to encourage acceptance of their use within the biologics cell line development space and provide guidance for regulatory submission using these alternative approaches.LAY ABSTRACT: In the manufacture of biologics produced in mammalian cells, one recommendation by regulatory agencies to help ensure product consistency, safety, and efficacy is to produce the material from a monoclonal cell line derived from a single, progenitor cell. The process by which monoclonality is assured can be supplemented with single-well plate images of the progenitor cell. Here we highlight the utility of that imaging technology, describe approaches to verify the validity of those images, and discuss how to analyze that information to support a biologic filing application. This approach serves as an industry perspective to increased regulatory interest within the scope of monoclonality for mammalian cell culture-derived biologics.

Selection of high expressing mammalian cells by surface display of reporters.

A flow cytometry method using a nonfluorescent reporter protein was developed for rapid, early-stage identification of cells producing high levels of a recombinant protein of interest. A cell surface reporter protein is coexpressed with the protein of interest, and the reporter protein is detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the protein of interest are linked by an IRES so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein's expression level accurately predicts, on a per cell basis, the relative expression level of the protein of interest. This method provides an effective process for selecting cells that express high levels of recombinant proteins, with the benefits of rapid and accurate 96-well plate clone screening (that is both quantitative and qualitative) and elimination of unstable clones during subsequent scale up and culture. Furthermore, because this method does not rely on the availability of a detection reagent specific for the protein of interest that is expressed, it can be easily implemented into any cell line development process.

Utilization of non-AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines.

Here we describe a method that couples flow cytometric detection with the attenuated translation of a reporter protein to enable efficient selection of CHO clones producing high levels of recombinant proteins. In this system, a small cell surface reporter protein is expressed from an upstream open reading frame utilizing a non-AUG initiation (alternate start) codon. Due to the low translation initiation efficiency of this alternate start codon, the majority of translation initiation events occur at the first AUG of the downstream open reading frame encoding the recombinant protein of interest. While translation of the reporter is significantly reduced, the levels are sufficient for detection using flow cytometric methods and, in turn, predictive of protein expression from the gene of interest since both ORFs are translated from the same mRNA. Using this system, CHO cells have been sorted to obtain enriched pools producing significantly higher levels of recombinant proteins than the starting cell population and clones with significantly better productivity than those generated from limiting dilution cloning. This method also serves as an effective screening tool during clone expansion to enable resources to be focused solely on clones with both high and stable expression.

Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen.

Flow cytometry was partnered with a nonfluorescent reporter protein for rapid, early stage identification of clones producing high levels of a therapeutic protein. A cell surface protein, not normally expressed on CHO cells, is coexpressed, as a reporter, with the therapeutic protein and detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the therapeutic protein are linked by an IRES, so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein's expression level accurately predicts the relative expression level of the therapeutic protein for each clone. This method provides an effective process for generating recombinant cell lines producing high levels of therapeutic proteins, with the benefits of rapid and accurate 96-well plate clone screening and elimination of unstable clones at an earlier stage in the development process. Furthermore, because this method does not rely on the availability of an antibody specific for the therapeutic protein being expressed, it can be easily implemented into any cell line development process.

The translation repressor 4E-BP2 is critical for eIF4F complex formation, synaptic plasticity, and memory in the hippocampus.

Long-lasting synaptic plasticity and memory requires mRNA translation, yet little is known as to how this process is regulated. To explore the role that the translation repressor 4E-BP2 plays in hippocampal long-term potentiation (LTP) and learning and memory, we examined 4E-BP2 knock-out mice. Interestingly, genetic elimination of 4E-BP2 converted early-phase LTP to late-phase LTP (L-LTP) in the Schaffer collateral pathway, likely as a result of increased eIF4F complex formation and translation initiation. A critical limit for activity-induced translation was revealed in the 4E-BP2 knock-out mice because L-LTP elicited by traditional stimulation paradigms was obstructed. Moreover, the 4E-BP2 knock-out mice also exhibited impaired spatial learning and memory and conditioned fear-associative memory deficits. These results suggest a crucial role for proper regulation of the eIF4F complex by 4E-BP2 during LTP and learning and memory in the mouse hippocampus.