Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells.

Introns are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to <0.5%. Subsequently, a comprehensive evaluation was conducted to understand the influence of the different introns in the HC genes on the expression of recombinant biotherapeutic antibodies. The data revealed an unexpected cooperation between specific introns for efficient splicing, where intron retention led to significant reductions in IgG expression of up to 75% for some intron combinations. Furthermore, it was shown that HC introns could be fully removed without significantly affecting productivity. This work paves the way for future biotherapeutic antibody transgene design with regard to inclusion of HC introns. By removing unnecessary introns, transgene mRNA transcript will no longer be mis-spliced, thereby eliminating HC splice variants and improving antibody product quality.

CHO synthetic promoters improve expression and product quality of biotherapeutic proteins.

When expressing complex biotherapeutic proteins, traditional expression plasmids and methods may not always yield sufficient levels of high-quality product. High-strength viral promoters commonly used for recombinant protein (rProtein) production in mammalian cells allow for maximal expression, but provide limited scope to alter their transcription dynamics. However, synthetic promoters designed to provide tunable transcriptional activity offer a plasmid engineering approach to more precisely regulate product quality, yield or to reduce product related contaminants. We substituted the viral promoter CMV with synthetic promoters that offer different transcriptional activities to express our gene of interest in Chinese hamster ovary (CHO) cells. Stable pools were established and the benefits of regulating transgene transcription on the quality of biotherapeutics were examined in stable pool fed-batch overgrow experiments. Specific control of gene expression of the heavy chain (HC):light chain (LC) of a Fab, and the ratio between the two HCs in a Duet mAb reduced levels of aberrant protein contaminants; and the controlled expression of the helper gene XBP-1s improved expression of a difficult-to-express mAb. This synthetic promoter technology benefits applications that require custom activity. Our work highlights the advantages of employing synthetic promoters for production of more complex rProteins.

Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells.

Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression- and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells.

Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters.

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.

A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies.

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 platform for context-specific genetic engineering of recombinant protein production by CHO 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.

Whole synthetic pathway engineering of recombinant protein production.

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.

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.

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

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.

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.

Febuxostat and Increased Dialysis as a Treatment for Severe Tophaceous Gout in a Hemodialysis Patient.

Uric acid accumulates in renal failure and is thought to be a uremic toxin-that is, higher levels of uric acid are more damaging to the kidneys. Urate crystals can precipitate in the kidney tubules, cause urate stones, and promote inflammatory changes in the renal interstitium and vascular endothelium. Uric acid is also a small non-protein-bound molecule and therefore easily dialyzable. Here, we present the case of an anuric hemodialysis patient with severe tophaceous gout who regained some renal function and whose gout burden significantly decreased resulting in marked improvement in functional status using a new gout medication, febuxostat, and increased frequency of dialysis.

Largemouth bass (Micropterus salmoides) and striped mullet (Mugil cephalus) as vectors of contaminants to human consumers in northwest Florida.

The health benefits of regular consumption of fish and seafood have been espoused for many years. However, fish are also a potential source of environmental contaminants that have well known adverse effects on human health. We investigated the consumption risks for largemouth bass (Micropterus salmoides; n = 104) and striped mullet (Mugil cephalus; n = 170), two commonly harvested and consumed fish species inhabiting fresh and estuarine waters in northwest Florida. Skinless fillets were analyzed for total mercury, inorganic arsenic, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/F), polychlorinated biphenyls (PCBs), and organochlorine pesticides. Contaminant levels were compared to screening values (SV) calculated using U.S. Environmental Protection Agency (EPA) recommendations for establishing consumption advisories. Largemouth bass were found to contain high levels of total mercury at all sampling locations (0.37-0.89 ug/g) and one location exhibited elevated total PCBs (39.4 ng/g). All of the samples exceeded Florida fish consumption advisory trigger levels for total mercury and one location exceeded the U.S. EPA SV for total PCBs. As a result of the high mercury levels, the non-cancer health risks (hazard index-HI) for bass were above 1 for all locations. Striped mullet from several locations with known point sources contained elevated levels of PCBs (overall range 3.4-59.3 ng/g). However, total mercury levels in mullet were low. Eight of the 16 mullet sampling locations exceeded the U.S. EPA SV for total PCBs and two locations exceeded an HI of 1 due to elevated PCBs. Despite the elevated levels of total PCBs in some samples, only two locations exceeded the acceptable cancer risk range and therefore cancer health risks from consumption of bass and mullet were determined to be low at most sampling locations.

Intradialytic hyperalimentation as adjuvant support in pregnant hemodialysis patients: case report and review of the literature.

Pregnancy in chronic dialysis patients is unusual and associated with many complications. Infants are often born both prematurely and small for gestational age. We report a case of a 36-year-old diabetic hemodialysis patient G4P3 who had prolonged hyperemesis gravidarum, for whom intradialytic parenteral nutrition (IDPN) was started at week 14 and continued throughout her pregnancy. She delivered a 3.5-kg baby girl at the 36th week of gestation by cesarean section. We discuss the use of IDPN as adjunct therapy for pregnant dialysis patients.

Increasing prosocial behavior and academic achievement among adolescent African American males.

African American adolescents disproportionately perform poorly compared to peers in both behavioral and academic aspects of their educational experience. In this study, African American male students participated in an after-school program involving tutoring, group counseling, and various enrichment activities. All students were assessed regarding their behavioral changes using attendance, discipline referrals, suspensions, and expulsions reports. The Kaufman Brief Intelligence Test (KBIT) and the Kaufman Test of Educational Achievement (KTEA) were used to assess the adolescents' improvement in their skills in reading and mathematics. After the end of the two-year program, initial results showed that the adolescents had increased their daily attendance, decreased discipline referrals, and had no suspensions or expulsions. These results also indicated that although the students entered the program at different skill levels, they were assessed to have the ability to function at their appropriate grade level. Their average improvement in basic skills was at least two grade levels. Implications drawn from the findings include: (a) there is a need to emphasize appropriate assessment prior to beginning a skill improvement program; (b) a need to emphasize the use of individualized learning plans and tutors; and (c) a need to further investigate the role of assessment and intervention in after-school programming in order to close the achievement gap.