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To successfully engineer mammalian cells for a desired purpose, multiple recombinant genes are required to be coexpressed at a specific and optimal ratio. In this study, we hypothesized that synthetic promoters varying in transcriptional activity could be used to create single multigene expression vectors coexpressing recombinant genes at a predictable relative stoichiometry. A library of 27 multigene constructs was created comprising three discrete fluorescent reporter gene transcriptional units in fixed series, each under the control of either a relatively low, medium, or high transcriptional strength synthetic promoter in every possible combination. Expression of each reporter gene was determined by absolute quantitation qRT-PCR in CHO cells. The synthetic promoters did generally function as designed within a multigene vector context; however, significant divergences from predicted promoter-mediated transcriptional activity were observed. First, expression of all three genes within a multigene vector was repressed at varying levels relative to coexpression of identical reporter genes on separate single gene vectors at equivalent gene copies. Second, gene positional effects were evident across all constructs where expression of the reporter genes in positions 2 and 3 was generally reduced relative to position 1. Finally, after accounting for general repression, synthetic promoter transcriptional activity within a local multigene vector format deviated from that expected. Taken together, our data reveal that mammalian synthetic promoters can be employed in vectors to mediate expression of multiple genes at predictable relative stoichiometries. However, empirical validation of functional performance is a necessary prerequisite, as vector and promoter design features can significantly impact performance.

The manufacture of bispecific antibodies by Chinese hamster ovary (CHO) cells is often hindered by lower product yields compared to monoclonal antibodies. Recently, reactive oxygen species have been shown to negatively impact antibody production. By contrast, strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression. With this in mind, we generated a novel hydrogen peroxide evolved host using directed host cell evolution. Here we demonstrate that this host has heritable resistance to hydrogen peroxide over many generations, displays enhanced antioxidant capacity through the upregulation of several, diverse antioxidant defense genes such as those involved in glutathione synthesis and turnover, and has improved glutathione content. Additionally, we show that this host has significantly improved transfection recovery times, improved growth and viability properties in a fed‐batch production process, and elevated expression of two industrially relevant difficult to express bispecific antibodies compared to unevolved CHO control host cells. These findings demonstrate that host cell evolution represents a powerful methodology for improving specific host cell characteristics that can positively impact the expression of difficult to express biotherapeutics., A novel H2O2 evolved CHO host was generated and characterised in this study. This host displays enhanced antioxidant capacity through the upregulation of several, diverse antioxidant defence genes such as those involved in glutathione synthesis and turnover and has improved glutathione content. Additionally, this host has significantly improved transfection recovery times, improved growth and viability properties in a fed batch production process and elevated expression of two industrially relevant difficult to express bispecific antibodies compared to unevolved CHO control host cells.

An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.

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.

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. © 2017 Wiley Periodicals, Inc.

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.

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.

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.