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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.