Diane Hatton

A high‐yielding CHO transient system: Coexpression of genes encoding EBNA‐1 and GS enhances transient protein expression

An efficient rapid protein expression system is crucial to support early drug development. Transient gene expression is an effective route, and to facilitate the use of the same host cells as for subsequent stable cell line development, we have created a high‐yielding Chinese hamster ovary (CHO) transient expression system. Suspension‐adapted CHO‐K1 host cells were engineered to express the gene encoding Epstein‐Barr virus (EBV) nuclear antigen‐1 (EBNA‐1) with and without the coexpression of the gene for glutamine synthetase (GS). Analysis of the transfectants indicated that coexpression of EBNA‐1 and GS enhanced transient expression of a recombinant antibody from a plasmid carrying an OriP DNA element compared to EBNA‐1‐only transfectants. This was confirmed with the retransfection of an EBNA‐1‐only cell line with a GS gene. The retransfected cell lines showed an increase in transient expression when compared with that of the EBNA‐1‐only parent. The transient expression process for the best CHO transient cell line was further developed to enhance protein expression and improve scalability by optimizing the transfection conditions and the cell culture process. This resulted in a scalable CHO transient expression system that is capable of expressing 2 g/L of recombinant proteins such as antibodies. This system can now rapidly provide gram amounts of recombinant antibody to supply preclinical development studies that has comparable product quality to antibody produced from a stably transfected CHO cell line.

N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains

Monoclonal antibodies (mAbs) contain short N‐terminal signal peptides on each individual polypeptide that comprises the mature antibody, targeting them for export from the cell in which they are produced. The signal peptide is cleaved from each heavy chain (Hc) and light chain (Lc) polypeptide after translocation to the ER and prior to secretion. This process is generally highly efficient, producing a high proportion of correctly cleaved Hc and Lc polypeptides. However, mis‐cleavage of the signal peptide can occur, resulting in truncation or elongation at the N‐terminus of the Hc or Lc. This is undesirable for antibody manufacturing as it can impact efficacy and can result in product heterogeneity. Here, we describe a truncated variant of the Lc that was detected during a routine developability assessment of the recombinant human IgG1 MEDI8490 in Chinese hamster ovary cells. We found that the truncation of the Lc was caused due to the use of the murine Hc signal peptide together with a lambda Lc containing an SYE amino acid motif at the N‐terminus. This truncation was not caused by mis‐processing of the mRNA encoding the Lc and was not dependent on expression platform (transient or stable), the scale of the fed‐batch culture or clonal lineage. We further show that using alternative signal peptides or engineering the Lc SYE N‐terminal motif prevented the truncation and that this strategy will improve Lc homogeneity of other SYE lambda Lc‐containing mAbs. Biotechnol. Bioeng. 2017;114: 1970–1977.

Transcriptome-based identification of the optimal reference CHO genes for normalisation of qPCR data

Real‐time quantitative PCR (qPCR) is the standard method for determination of relative changes in mRNA transcript abundance. Analytical accuracy, precision and reliability are critically dependent on the selection of internal control reference genes. In this study, the authors have identified optimal reference genes that can be utilised universally for qPCR analysis of CHO cell mRNAs. Initially, transcriptomic datasets were analysed to identify eight endogenous genes that exhibited high expression stability across four distinct CHO cell lines sampled in different culture phases. The relative transcript abundance of each gene in 20 diverse, commonly applied experimental conditions was then determined by qPCR analysis. Utilizing GeNorm, BestKeeper and NormFinder algorithms, the authors identified four mRNAs (Gnb1, Fkbp1a, Tmed2 and Mmadhc) that exhibited a highly stable level of expression across all conditions, validating their utility as universally applicable reference genes. Whilst any combination of only two genes can be generally used for normalisation of qPCR data, the authors show that specific combinations of reference genes are particularly suited to discrete experimental conditions. In summary, the authors report the identification of fully validated universal reference genes, optimised primer sequences robust to genomic mutations and simple reference gene pair selection guidelines that enable streamlined qPCR analyses of mRNA abundance in CHO cells with maximum accuracy and precision.

High-Yielding CHO Cell Pools for Rapid Production of Recombinant Antibodies

Cambridge Antibody Technology (CAT) has developed a system using GS-CHO transfectant pools to rapidly produce gram amounts of multiple IgGs for early characterisation studies and expedite drug development. The system involves screening a small number of independent pools by assessment of IgG harvest titre from terminal cultures or by flow cytometric analyses of intracellular IgG, which allows a more rapid ranking of pool performance. The highest-yielding pools are then expanded for production and can express up to 1.4 g/L at 5 L bioreactor scale in 7.5 weeks from transfection. Other GS-CHO transfectant pools have been scaled up to 50 L in disposable wavebags, and pools have been shown to be suitable for scale-up beyond 100 L, allowing the rapid production of tens of grams of IgG. The pools and manufacturing clonal cell lines at CAT use the same host cell type, expression system and production process therefore minimising the potential for differences in product characteristics at different stages of drug development. Productive pools have also been cloned out to identify high-yielding cell lines that show similar productivities to more conventionally isolated clonal cell lines, thereby potentially efficiently integrating rapid supply of antibody for early testing with manufacturing cell line development.

Engineering the expression of an anti-interleukin-13 antibody through rational design and mutagenesis

High levels of protein expression are key to the successful development and manufacture of a therapeutic antibody. Here, we describe two related antibodies, Ab001 and Ab008, where Ab001 shows a markedly lower level of expression relative to Ab008 when stably expressed in Chinese hamster ovary cells. We use single-gene expression vectors and structural analysis to show that the reduced titer is associated with the VL CDR2 of Ab001. We adopted two approaches to improve the expression of Ab001. First, we used mutagenesis to change single amino-acid residues in the Ab001 VL back to the equivalent Ab008 residues but this resulted in limited improvements in expression. In contrast when we used an in silico structure-based design approach to generate a set of five individual single-point variants in a discrete region of the VL, all exhibited significantly improved expression relative to Ab001. The most successful of these, D53N, exhibited a 25-fold increase in stable transfectants relative to Ab001. The functional potency of these VL-modified antibodies was unaffected. We expect that this in silico engineering strategy can be used to improve the expression of other antibodies and proteins.

New Mammalian Expression Systems

There are an increasing number of recombinant antibodies and proteins in preclinical and clinical development for therapeutic applications. Mammalian expression systems are key to enabling the production of these molecules, and Chinese hamster ovary (CHO) cell platforms continue to be central to delivery of the stable cell lines required for large-scale production. Increasing pressure on timelines and efficiency, further innovation of molecular formats and the shift to new production systems are driving developments of these CHO cell line platforms. The availability of genome and transcriptome data coupled with advancing gene editing tools are increasing the ability to design and engineer CHO cell lines to meet these challenges. This chapter aims to give an overview of the developments in CHO expression systems and some of the associated technologies over the past few years.

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies

ABSTRACT The conserved glycosylation site Asn297 of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX™ Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.

Whole synthetic pathway engineering of recombinant protein production: BROWN et al.

The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass. In this study, we describe how these two properties can be simultaneously optimized via design of a product‐specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10‐fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory’s capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette–chassis combinations should be applicable to diverse recombinant protein and host cell‐type contexts.

Control of amino acid transport into Chinese hamster ovary cells: GEOGHEGAN et al.

Amino acid transporters (AATs) represent a key interface between the cell and its environment, critical for all cellular processes: Energy generation, redox control, and synthesis of cell and product biomass. However, very little is known about the activity of different functional classes of AATs in Chinese hamster ovary (CHO) cells, how they support cell growth and productivity, and the potential for engineering their activity and/or the composition of amino acids in growth media to improve CHO cell performance in vitro. In this study, we have comparatively characterized AAT expression in untransfected and monoclonal antibody (MAb)‐producing CHO cells using transcriptome analysis by RNA‐seq, and mechanistically dissected AAT function using a variety of transporter‐specific chemical inhibitors, comparing their effect on cell proliferation, recombinant protein production, and amino acid transport. Of a possible 56 mammalian plasma membrane AATs, 16 AAT messenger RNAs (mRNAs) were relatively abundant across all CHO cell populations. Of these, a subset of nine AAT mRNAs were more abundant in CHO cells engineered to produce a recombinant MAb. Together, upregulated AATs provide additional supply of specific amino acids overrepresented in MAb biomass compared to CHO host cell biomass, enable transport of synthetic substrates for glutathione synthesis, facilitate transport of essential amino acids to maintain active protein synthesis, and provide amino acid substrates for coordinated antiport systems to maintain supplies of proteinogenic and essential amino acids.

In silico design of context-responsive mammalian promoters with user-defined functionality

Abstract Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities. As TFRE order and spacing did not affect the performance of these TFRE-combinations, compositions could be specifically arranged to preclude the formation of undesirable sequence features. This facilitated simple in silico-design of promoters with context-required, user-defined functionalities. To demonstrate this, we de novo-created promoters for biopharmaceutical production in CHO cells that exhibited precisely designed activity dynamics and long-term expression-stability, without causing observable retroactive effects on cellular performance. The design process described can be utilized for applications requiring context-responsive, customizable promoter function, particularly where co-expression of synthetic TFs is not suitable. Although the synthetic promoter structure utilized does not closely resemble native mammalian architectures, our findings also provide additional support for a flexible billboard model of promoter regulation.