Search

Environmental growth-inhibition conditions (GICs) have been used extensively for increasing cell-specific productivity (q P ) of Chinese hamster ovary (CHO) cells, with the most common being temperature downshift and sodium butyrate (NaBu) treatment. B lymphocyte-induced maturation protein-1 (BLIMP1) overexpression in CHO cells can also inhibit cell growth and increase product titers and q P . Given the similar responses, this study evaluated the individual and combined effects of BLIMP1 expression, low temperature, and NaBu treatment on culture performance, cell metabolism, and recombinant protein production of CHO cells. As expected, all three interventions decreased cell growth, arrested cells in G1/G0 cell cycle phase, and increased q P . However, CHO cells presented different responses when considering cell viability, recombinant gene expression, and cell metabolism that indicated differences in the molecular loci by which BLIMP1 and GICs generated higher productivities. Combinations of BLIMP1 expression and GICs acted synergistically to inhibit cell growth and maximize r-protein production, with the BLIMP1/NaBu condition leading to the most significant improvements in product titers and q P . This latter condition also proved to substantially increase product yields (up to 9.8 g immunoglobulin G1 [IgG1]/L and 2.2 g erythropoietin-Fc [EPO-Fc]/L) and q P (up to 179 pg/cell/day [pcd] for IgG1 and 30 pcd for EPO-Fc) in high-density perfusion cultures. These findings offered mechanistic insights about the productivity-enhancing effects of BLIMP1 and GICs, as well as their complementarity for generating highly productive processes., (© 2021 Wiley Periodicals LLC.)

This paper presents a straightforward approach for measuring and quantifying orthogonality directly in complex cell culture fluids (CCFs) without the requirement for tracking the retention behaviors of large sets of proteins. Null-producing CCFs were fractionated using linear salt gradients at constant pH on a set of multimodal resins. Fractions were then analyzed by ultraperformance-reversed phase liquid chromatography and the resulting chromatograms provided host cell protein (HCP) "fingerprints." Using these fingerprints, an inner product vector-based approach was employed to quantify the degree of orthogonality between pairs of resins and operating conditions for these large HCP protein sets. To compare resin orthogonality behavior in different expression systems, the Chinese hamster ovary and Pichia pastoris null-producing CCFs were examined. Orthogonality in multimodal systems was found to strongly depend on the expression system and the HCPs being screened. We also identified several unexpected pairs of multimodal resins within the same family that exhibited significant orthogonality. Furthermore, "self-orthogonality" was evaluated between resins operated at different pHs, and important operating regimes were identified for maximizing orthogonal selectivities. The framework developed in this paper for calculating orthogonality without the need for labor-intensive HCP tracking has important implications for efficient process development and resin/operating condition selection for both monoclonal antibody (mAb) polishing steps and non-mAb processes. In addition, this study provides a tool to unlock the untapped potential of multimodal resins by aiding in their rational selection and incorporation. Finally, the orthogonality framework here can facilitate the development of sets of next-generation multimodal resins specifically designed to provide highly orthogonal and efficient separations tailored for different expression systems., (© 2021 Wiley Periodicals LLC.)

Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small- and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Process conditions established during the development and manufacture of recombinant protein therapeutics dramatically impacts their quality and clinical efficacy. Technologies that enable rapid assessment of product quality are critically important. Here, we describe the development of sensor interfaces that directly connect to electronics and enable near real-time assessment of antibody titer and N-linked galactosylation. We make use of a spatially resolved electroassembled thiolated polyethylene glycol hydrogel that enables electroactivated disulfide linkages. For titer assessment, we constructed a cysteinylated protein G that can be linked to the thiolated hydrogel allowing for robust capture and assessment of antibody concentration. For detecting galactosylation, the hydrogel is linked with thiolated sugars and their corresponding lectins, which enables antibody capture based on glycan pattern. Importantly, we demonstrate linear assessment of total antibody concentration over an industrially relevant range and the selective capture and quantification of antibodies with terminal β-galactose glycans. We also show that the interfaces can be reused after surface regeneration using a low pH buffer. Our functionalized interfaces offer advantages in their simplicity, rapid assembly, connectivity to electronics, and reusability. As they assemble directly onto electrodes that also serve as I/O registers, we envision incorporation into diagnostic platforms including those in manufacturing settings., (© 2021 Wiley Periodicals LLC.)

Protein quantification during bioprocess monitoring is essential for biopharmaceutical manufacturing and is complicated by the complex chemical composition of the bioreactor broth. Here we present the early-stage development and optimization of a polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a hydrolysate-protein model (mimics clarified bioreactor broth samples) using a standard benchtop laboratory fluorometer. We used UV transmitting polarizers to provide wider range pTSFS spectra for screening of the four different TSFS spectra generated by the measurement: parallel (||), perpendicular (⊥), unpolarized (T) intensity spectra and anisotropy maps. TSFS || (parallel polarized) measurements were the best for protein quantification compared to standard unpolarized measurements and the Bradford assay. This was because TSFS || spectra had a better analyte signal to noise ratio (SNR), due to the anisotropy of protein emission. This meant that protein signals were better resolved from the background emission of small molecule fluorophores in the cell culture media. SNR of >5000 was achieved for concentrations of bovine serum albumin/yeastolate 1.2/10 g L -1 with TSFS || . Optimization using genetic algorithm and interval partial least squares based variable selection enabled reduction of spectral resolution and number of excitation wavelengths required without degrading performance. This enables fast (<3.5 min) online/at-line measurements, and the method had an LOD of 0.18 g L -1 and high accuracy with a predictive error of <9%., (© 2021 Wiley Periodicals LLC.)

The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy ("self-eating") is a cellular process that allows cells to mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy-inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed-batch serum-free cultures producing a human Immunoglobulin G1 (IgG1). Interestingly, the addition of 1-4 µM AIP enhanced productivity in a concentration-dependent manner. Cell-specific productivity increased up to 1.8-fold in batch cultures, while in fed-batch cultures a maximum twofold increase in IgG concentration was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields., (© 2021 Wiley Periodicals LLC.)

A continuous Chinese hamster ovary (CHO) cell culture process comprised of a highly proliferative N-1 perfusion bioreactor utilizing a hydrocyclone as a cell retention device linked to a production continuous-flow stirred tank reactor (CSTR) is presented. The overflow stream from the hydrocyclone, which is only partially depleted of cells, provides a continuous source of high viability cells from the N-1 perfusion bioreactor to the 5-20 times larger CSTR. Under steady-state conditions, this linked-bioreactor system achieved a peak volumetric productivity of 0.96 g/L/day, twofold higher than the optimized fed-batch process. The linked bioreactor system using a hydrocyclone was also shown to be 1.8-3.1 times more productive than a dual, cascading CSTR system without cell retention., (© 2021 Wiley Periodicals LLC.)

The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5'-adenosyl)- l-methionine (SAM) and 5'-deoxy-5'-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti-IL-8-producing CHO-DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells., (© 2020 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC.)

Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) operation during an initial scale-up run for an immunoglobulin-G 4 (IgG4) monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high-molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50-200 L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This study demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale., (© 2020 Wiley Periodicals LLC.)

Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach., (© 2020 Wiley Periodicals LLC.)

Recombinant protein production (RPP) in Escherichia coli (E. coli) often induces metabolic burden to the cells that compromise their overall growth and productivity. Amino acid starvation due to RPP is a major contributor of the metabolic burden on the cells and induces global stress response known as a stringent-like response. In this study, the effect of amino acid supplementation in a chemically defined medium on cellular growth and recombinant pramlintide production was investigated. Based on the consumption profile, few amino acids were categorized as growth-promoting (GP1) and protein production promoting (GP2). Feeding strategies of GP1 and GP2 were tested in shake flasks followed by scale up into the bioreactor. A 40% increase in the recombinant pramlintide (rPramlintide) production (protein concentration of 3.09 ± 0.12 g/L and yield of 227.69 ± 19.72 mg pramlintide per gram dry cell weight) was realized. Furthermore, transcriptomics data indicated the downregulation of several genes associated with global stress response and genes involved in amino acid biosynthesis in test culture, supported by proteomics analysis. These results signify that the external supply of critical amino acids decreases cellular stress during RPP and improves process productivity., (© 2020 Wiley Periodicals LLC.)

The bacterium Escherichia coli is among the most popular hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently generated the specialized MP-producing E. coli strain SuptoxD, which upon co-expression of the effector gene djlA, is capable of alleviating two major bottlenecks in bacterial recombinant MP production: it suppresses the toxicity that frequently accompanies the MP-overexpression process and it markedly increases the cellular accumulation of membrane incorporated and properly folded recombinant MP. Combined, these two positive effects result in dramatically enhanced volumetric yields for various recombinant MPs of both prokaryotic and eukaryotic origin. Based on the observation that djlA is found in the genomes of various pathogenic bacteria, the aim of the present work was to investigate (a) whether other naturally occurring DjlA variants can exert the MP toxicity-suppressing and production-promoting effects similarly to the E. coli DjlA and (b) if we can identify a DjlA variant whose efficiency surpasses that of the E. coli DjlA of SuptoxD. We report that a quite surprisingly broad variety of homologous DjlA proteins exert beneficial effects on recombinant MP when overexpressed in E. coli. Furthermore, we demonstrate that the Salmonella enterica DjlA is an even more potent enhancer of MP productivity compared with the E. coli DjlA of SuptoxD. Based on this, we constructed a second-generation SuptoxD strain, termed SuptoxD2.0, whose MP-production capabilities surpass significantly those of the original SuptoxD, and we anticipate that SuptoxD2.0 will become a broadly utilized expression host for recombinant MP production in bacteria., (© 2020 Wiley Periodicals LLC.)

Packaging during the passaging of viruses in cell cultures yields various phenotypes and is regulated by viral protein expression in infected cells. Although such a packaging mechanism has a profound effect in controlling the virus yield, little is known about the underlying statistical models followed by virus packaging and protein expression among cells infected with the virus. A predictive framework combining identification of the probability density function (PDF) based on log-likelihood and using the PDF for Monte-Carlo simulations is developed. The Birnbaum-Saunders distribution was found to be consistent with all three-virus packaging levels, including nucleocapsids/occlusion-derived virus (ODV), ODVs/polyhedra, and polyhedra/cell for both wild-type and genetically modified AcMNPV. Next, it was demonstrated that PDF fitting could be used to compare two viruses having distinctly different genetic configurations. Finally, the identified PDF can be incorporated in RNA synthesis parameters for baculovirus infection to predict the cell-to-cell variability in protein expression using Monte-Carlo simulations. The proposed tool can be used for the estimation of uncertainty in the kinetic parameter and prediction of cell-to-cell variability for other biological systems., (© 2020 Wiley Periodicals, Inc.)

Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy-to-express antibody, whereas Erp27 and Erp57 co-overexpression increased cell density, viability, and the yield of difficult-to-express proteins. Foxa1 overexpression increased cell density, cell viability, and easy- and difficult-to-express protein yields, whereas it decreased reactive oxygen species late in fed-batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult- and/or easy-to-express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase-activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein-expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by-products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

Protein translation has emerged as a critical bottleneck for overall productivity of biological molecules. An augmentation of protein translation can be achieved by cell line engineering or by sophisticated vector design. However, for industrial process development purposes, identification of media additives that promote translation will be of great value, obviating the generation of new host platforms. Here, we examined the effect of low cadmium chloride concentrations on protein synthesis and cell line productivity. At low micromolar concentrations, cadmium chloride induced the mTOR pathway and promoted total protein synthesis in HEK 293T and CHO-K1 cells with minimal toxicity. In a parallel screening of kinase inhibitors for promoting protein expression, we identified the RSK1 inhibitor, BI-D1870, as having a transcription promoting activity on cytomegalovirus promoter-driven transgenes. Fed-batch analyses of CHO-K1 cells producing the anticoagulant factor tissue plasminogen activator (tPA) demonstrated that inclusion of cadmium chloride alone and particularly in combination with BI-D1870 improved overall yields of tPA by more than two-fold with minimal effect on cell growth. We, therefore, underscore the use of cadmium alone and in combination with BI-D1870 for improving bioproduction yields., (© 2018 Wiley Periodicals, Inc.)

Recombinant CHO (Chinese hamster ovary) cell lines producing therapeutic proteins often lose their production capability during long-term cultivation. To ensure that CHO production cell lines can be up-scaled to high-volume bioreactors, labor intensive stability studies of several months have to be performed to deselect clones that are losing productivity over time. The ability to predict whether clones will produce recombinant proteins at constant high levels, for example, through determination of biomarkers such as expression of specific genes, plasmid integration sites, or epigenetic patterns, or even to improve CHO host cell lines to increase the probability of the generation of stable clones would be highly beneficial. Previously, we reported that the lack of a telomeric region of chromosome 8 correlates with increased productivities and higher production stabilities of monoclonal antibody expressing CHO cell lines (Ritter A, Voedisch B, Wienberg J, Wilms B, Geisse S, Jostock T, Laux H. 2016a. Biotechnol Bioeng 113(5):1084-1093). Herein, we describe that the knock-out of the gene Fam60A, which is one of the genes located within the telomeric region of chromosome 8, in CHO-K1a cells leads to the isolation of significantly more clones with higher protein production stabilities of monoclonal antibodies during long-term cultivation. Biotechnol. Bioeng. 2017;114: 701-704. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The load phase in preparative Protein A capture steps is commonly not controlled in real-time. The load volume is generally based on an offline quantification of the monoclonal antibody (mAb) prior to loading and on a conservative column capacity determined by resin-life time studies. While this results in a reduced productivity in batch mode, the bottleneck of suitable real-time analytics has to be overcome in order to enable continuous mAb purification. In this study, Partial Least Squares Regression (PLS) modeling on UV/Vis absorption spectra was applied to quantify mAb in the effluent of a Protein A capture step during the load phase. A PLS model based on several breakthrough curves with variable mAb titers in the HCCF was successfully calibrated. The PLS model predicted the mAb concentrations in the effluent of a validation experiment with a root mean square error (RMSE) of 0.06 mg/mL. The information was applied to automatically terminate the load phase, when a product breakthrough of 1.5 mg/mL was reached. In a second part of the study, the sensitivity of the method was further increased by only considering small mAb concentrations in the calibration and by subtracting an impurity background signal. The resulting PLS model exhibited a RMSE of prediction of 0.01 mg/mL and was successfully applied to terminate the load phase, when a product breakthrough of 0.15 mg/mL was achieved. The proposed method has hence potential for the real-time monitoring and control of capture steps at large scale production. This might enhance the resin capacity utilization, eliminate time-consuming offline analytics, and contribute to the realization of continuous processing. Biotechnol. Bioeng. 2017;114: 368-373. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc., (© 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

Continuous manufacturing is currently being seriously considered in the biopharmaceutical industry as the possible new paradigm for producing therapeutic proteins, due to production cost and product quality related benefits. In this study, a monoclonal antibody producing CHO cell line was cultured in perfusion mode and connected to a continuous affinity capture step. The reliable and stable integration of the two systems was enabled by suitable control loops, regulating the continuous volumetric flow and adapting the operating conditions of the capture process. For the latter, an at-line HPLC measurement of the harvest concentration subsequent to the bioreactor was combined with a mechanistic model of the capture chromatographic unit. Thereby, optimal buffer consumption and productivity throughout the process was realized while always maintaining a yield above the target value of 99%. Stable operation was achieved at three consecutive viable cell density set points (20, 60, and 40 × 10 6 cells/mL), together with consistent product quality in terms of aggregates, fragments, charge isoforms, and N-linked glycosylation. In addition, different values for these product quality attributes such as N-linked glycosylation, charge variants, and aggregate content were measured at the different steady states. As expected, the amount of released DNA and HCP was significantly reduced by the capture step for all considered upstream operating conditions. This study is exemplary for the potential of enhancing product quality control and modulation by integrated continuous manufacturing. Biotechnol. Bioeng. 2017;114: 298-307. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc., (© 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)

The use of biological systems to synthesize complex therapeutic products has been a remarkable success. However, during product development, great attention must be devoted to defining acceptable levels of impurities that derive from that biological system, heading this list are host cell proteins (HCPs). Recent advances in proteomic analytics have shown how diverse this class of impurities is; as such knowledge and capability grows inevitable questions have arisen about how thorough current approaches to measuring HCPs are. The fundamental issue is how to adequately measure (and in turn monitor and control) such a large number of protein species (potentially thousands of components) to ensure safe and efficacious products. A rather elegant solution is to use an immunoassay (enzyme-linked immunosorbent assay [ELISA]) based on polyclonal antibodies raised to the host cell (biological system) used to synthesize a particular therapeutic product. However, the measurement is entirely dependent on the antibody serum used, which dictates the sensitivity of the assay and the degree of coverage of the HCP spectrum. It provides one summed analog value for HCP amount; a positive if all HCP components can be considered equal, a negative in the more likely event one associates greater risk with certain components of the HCP proteome. In a thorough risk-based approach, one would wish to be able to account for this. These issues have led to the investigation of orthogonal analytical methods; most prominently mass spectrometry. These techniques can potentially both identify and quantify HCPs. The ability to measure and monitor thousands of proteins proportionally increases the amount of data acquired. Significant benefits exist if the information can be used to determine critical HCPs and thereby create an improved basis for risk management. We describe a nascent approach to risk assessment of HCPs based upon such data, drawing attention to timeliness in relation to biosimilar initiatives. The development of such an approach requires databases based on cumulative knowledge of multiple risk factors that would require national and international regulators, standards authorities (e.g., NIST and NIBSC), industry and academia to all be involved in shaping what is the best approach to the adoption of the latest bioanalytical technology to this area, which is vital to delivering safe efficacious biological medicines of all types., (© 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)

The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system., (© 2015 Wiley Periodicals, Inc.)

Saccharomyces cerevisiae is a useful platform for protein production of biopharmaceuticals and industrial enzymes. To date, substantial effort has focused on alleviating several bottlenecks in expression and the secretory pathway. Recently, it has been shown that highly active endocytosis could decrease the overall protein titer in the supernatant. In this study, we block endocytosis and trafficking to the vacuole using a modified TEV Protease-Mediated Induction of Protein Instability (mTIPI) system to disrupt the endocytotic and vacuolar complexes. We report that conditional knock-down of endocytosis gene Rvs161 improved the concentration of α-amylase in supernatant of S. cerevisiae cultures by 63.7% compared to controls. By adaptive evolution, we obtained knock-down mutants in Rvs161 and End3 genes with 2-fold and 3-fold α-amylase concentrations compared to controls that were not evolved. Our study demonstrates that genetic blocking of endocytotic mechanisms can improve heterologous protein production in S. cerevisiae. This result is likely generalizable to other eukaryotic secretion hosts., (© 2014 Wiley Periodicals, Inc.)

Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium., (© 2014 Wiley Periodicals, Inc.)

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures., (© 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)

Multi-wavelength fluorescence spectroscopy was investigated as a potential tool for use in monitoring key process variables that include: viable and dead cells, recombinant protein, glucose, and ammonia concentrations for Chinese hamster ovary (CHO) cells during cultivation.For the purpose of calibrating the fluorescence-based empirical model, cells were grown in batch mode with different initial glucose and glutamine concentrations.Spectrofluorometer settings were optimized to ensure reproducibility and accuracy of the acquired spectra. With the purpose of gaining qualitative insight into the evolution of the spectra, the trajectories of individual fluorophore peaks were studied during the cultivation process. Spectral changes related to biomass and secreted proteins were investigated by comparing the spectra at various stages during the downstream processing. A partial least square regression (PLSR) was used to formulate empirical models that related the input data set, i.e., the fluorescence excitation-emission matrix, to the actual state of the system including viable cell and dead cells and recombinant protein, glucose, and ammonia concentrations. The models exhibited accurate prediction ability for the process variables of interest.

Although the molecular mechanisms by which host cells defend themselves against viral infection have been studied in great depth, and countermeasures viruses employ to suppress such defensive responses have been widely documented, relatively little attention has been devoted toward elucidating how such interactions between virus and host are resolved over multiple rounds of infection. Here, we describe the design, synthesis, and validation of a dual-color fluorescent reporter system to study how viral infections spread through a host cell monolayer and how the cellular innate immune system mounts an antiviral response. We employed recombinant, red fluorescent protein expressing mutants of a prototypical RNA virus, vesicular stomatitis virus to enable identification and tracking of infected cells. Further, we generated stable reporter cells that use green fluorescent protein to report on the expression of IFIT2, an interferon stimulated gene involved in the interference of viral protein translation, and a marker of antiviral defense activation. The presence of the fluorescent protein reporters had minimal effects on the normal behavior of the cells or viruses. Moreover, expression of the virus and cell reporters correlated with the kinetics of viral replication and activation of an anti-viral response, respectively. This two-color system enabled us to track and quantify in live cells how viral replication and activation of host defensive responses play out over multiple rounds of infection. Initial study of propagating infections demonstrated that antiviral activation over multiple rounds was critical for slowing and ultimately halting the spread of infection., (© 2013 Wiley Periodicals, Inc.)

Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest that productivity is rather a minor feature in the context of global transcriptional or protein expression space., (© 2013 Wiley Periodicals, Inc.)

Baculovirus infection boosts the host biosynthetic activity towards the production of viral components and the recombinant protein of interest, hyper-productive phenotypes being the result of a successful adaptation of the cellular network to that scenario. Spodoptera frugiperda derived Sf9 and Trichoplusia ni derived High Five cell lines have a major track record for the production of recombinant proteins, with High Five cells presenting higher productivities. A metabolic profiling of the two insect cell lines was pursued to underpin specific cellular traits behind productive phenotypes. Multivariate analysis identified cell-line dependent metabolic signatures linked to productivity. Pathway analysis highlighted cellular pathways of paramount importance in supporting infection and protein production. Moreover, better producer phenotypes proved to be correlated with the capacity of cells to shift their metabolism in favor of energy-generating pathways to fuel biosynthesis, a scenario observed in the High Five cell line. Metabolomic profiling allowed us to identify metabolic pathways involved in infection and recombinant protein production, which can be selected as targets for further improvement of the system., (© 2013 Wiley Periodicals, Inc.)

Since detailed evaluation of specific transglutaminases (TGs) from various species requires identification of their substrate specificities, rapid substrate screening method by measurement of their relative activities is in great demand. Here, a novel evaluation method of TG activity was developed using two recombinant fluorescent proteins (FPs), that is, eYFP and DsRed, tagged with TG substrate peptides. By cross-linking the two FPs based on the tagged target peptide sequences at their C-terminus, the expression of co-transformed TG allows quenching of the yellow fluorescence intensities. It was shown that the degree of in vivo fluorescent quenching by the TG activity agrees well with its in vitro reaction data, suggesting that this system can be used to identify relative substrate specificity of TGs for target peptide sequences. Using this method, the lysine substrates of TGs from Bacillus species (BTG) were evaluated, and the newly selected pentapeptide, KTKTN showed almost the same reactivity with the well-known hexa-lysine (K₆) substrate for BTG reaction., (© 2013 Wiley Periodicals, Inc.)

Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7 ± 0.5 µg mL(-1) with batch reactions lasting up to 2 h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (µg protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from ∼2 × 10(-4) to ∼4 × 10(-1)  µg $(-1) , which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries., (Copyright © 2013 Wiley Periodicals, Inc.)

High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30 g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation., (Copyright © 2013 Wiley Periodicals, Inc.)

Two kinds of disposable bioreactors, air-lift disposable bioreactors (ADB) and wave disposable bioreactors (WDB) were compared with stirred-tank reactors (5-L STR). These bioreactors were successfully applied to transgenic rice cell cultures for the production of recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). In both systems, a fed-batch culture method was used to produce hCTLA4Ig efficiently by feeding concentrated amino acids and production levels were enhanced when dissolved oxygen (DO) level was regulated at 30% using pure oxygen sparging. Agitation and aeration rate during cultivation in ADB and WDB were determined by the same mixing time. The results in both disposable bioreactors showed similar values in maximum cell density (11.9 gDCW/L and 12.6 gDCW/L), doubling time (4.8- and 5.0-day), and maximum hCTLA4Ig concentration (43.7 and 43.3 mg/L). Relatively higher cell viability was sustained in the ADB whereas hCTLA4Ig productivity was 1.2-fold higher than that in WDB. The productivity was improved by increasing aeration rate (0.2 vvm). Overall, our experiments demonstrate pneumatically driven disposable bioreactors are applicable for the production of recombinant proteins in plant cell cultures. These results will be useful for development and scale-up studies of disposable bioreactor systems for transgenic plant cell cultures., (Copyright © 2013 Wiley Periodicals, Inc.)

Chinese hamster ovary (CHO) cells are commonly used for industrial production of recombinant proteins in fed batch or alternative production systems. Cells progress through multiple metabolic stages during fed-batch antibody (mAb) production, including an exponential growth phase accompanied by lactate production, a low growth, or stationary phase when specific mAb production increases, and a decline when cell viability declines. Although media composition and cell lineage have been shown to impact growth and productivity, little is known about the metabolic changes at a molecular level. Better understanding of cellular metabolism will aid in identifying targets for genetic and metabolic engineering to optimize bioprocess and cell engineering. We studied a high expressing recombinant CHO cell line, designated high performer (HP), in fed-batch productions using stable isotope tracers and biochemical methods to determine changes in central metabolism that accompany growth and mAb production. We also compared and contrasted results from HP to a high lactate producing cell line that exhibits poor growth and productivity, designated low performer (LP), to determine intrinsic metabolic profiles linked to their respective phenotypes. Our results reveal alternative metabolic and regulatory pathways for lactate and TCA metabolite production to those reported in the literature. The distribution of key media components into glycolysis, TCA cycle, lactate production, and biosynthetic pathways was shown to shift dramatically between exponential growth and stationary (production) phases. We determined that glutamine is both utilized more efficiently than glucose for anaplerotic replenishment and contributes more significantly to lactate production during the exponential phase. Cells shifted to glucose utilization in the TCA cycle as growth rate decreased. The magnitude of this metabolic switch is important for attaining high viable cell mass and antibody titers. We also found that phosphoenolpyruvate carboxykinase (PEPCK1) and pyruvate kinase (PK) are subject to differential regulation during exponential and stationary phases. The concomitant shifts in enzyme expression and metabolite utilization profiles shed light on the regulatory links between cell metabolism, media metabolites, and cell growth., (Copyright © 2013 Wiley Periodicals, Inc.)

Live-cell assays to measure cellular function performed within 3D cultures have the potential to elucidate the underlying processes behind disease progression and tissue formation. Cells cultured in 3D interact and remodel their microenvironment and can develop into complex structures. We have developed a transcription factor (TF) activity array that uses bioluminescence imaging (BLI) of lentiviral delivered luminescent reporter constructs that allows for the non-invasive imaging of TF activity in both 2D and 3D culture. Imaging can be applied repeatedly throughout culture to capture dynamic TF activity, though appropriate normalization is necessary. We investigated in-well normalization using Gaussia or Renilla luciferase, and external well normalization using firefly luciferase. Gaussia and Renilla luciferase were each unable to provide consistent normalization for long-term measurement of TF activity. However, external well normalization provided low variability and accounted for changes in cellular dynamics. Using external normalization, dynamic TF activities were quantified for five TFs. The array captured expected changes in TF activity to stimuli, however the array also provided dynamic profiles within 2D and 3D that have not been previously characterized. The development of the technology to dynamically track TF activity within cells cultured in both 2D and 3D can provide greater understanding of complex cellular processes., (Copyright © 2012 Wiley Periodicals, Inc.)

In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing--new parental cell lines which are inherently more "fit for purpose." One-hundred and ninety-nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell-specific proliferation rate during extended deep-well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell-specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub-culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N-glycan processing were identified. Finally, for clonal populations most "evolved" by extended sub-culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N-glycosylation. Rapid-specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone-specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and isolate CHO cell variants with improved functional properties in vitro., (Copyright © 2012 Wiley Periodicals, Inc.)

Host cell engineering is becoming a realistic option in whole bioprocess strategies to maximize product manufacturability. High molecular weight (MW) genomic DNA currently hinders bioprocessing of Escherichia coli by causing viscosity in homogenate feedstocks. We previously showed that co-expressing Staphylococcal nuclease and human Fab' fragment in the periplasm of E. coli enables auto-hydrolysis of genomic DNA upon cell disruption, with a consequent reduction in feedstock viscosity and improvement in clarification performance. Here we report the impact of periplasmic nuclease expression on stability of DNA and Fab' fragment in homogenates, host-strain growth kinetics, cell integrity at harvest and Fab' fragment productivity. Nuclease and Fab' plasmids were shown to exert comparable levels of growth burden on the host W3110 E. coli strain. Nuclease co-expression did not compromise either the growth performance or volumetric yield of the production strain. 0.5 g/L Fab' fragment (75 L scale) and 0.7 g/L (20 L scale) was achieved for both unmodified and cell-engineered production strains. Unexpectedly, nuclease-modified cells achieved maximum Fab' levels 8-10 h earlier than the original, unmodified production strain. Scale-down studies of homogenates showed that nuclease-mediated hydrolysis of high MW DNA progressed to completion within minutes of homogenization, even when homogenates were chilled on ice, with no loss of Fab' product and no need for additional co-factors or buffering., (Copyright © 2011 Wiley Periodicals, Inc.)

In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG(4) monoclonal antibody production by GS-CHO cells throughout a state-of-the-art industrial fed-batch culture process. Based on the calculation of a production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy-intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of production. Instead, cell-specific Mab production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole-process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase production of a recombinant protein by a mammalian host cell--where control of productivity is inherently protein product and cell line specific., (Copyright © 2011 Wiley Periodicals, Inc.)

With the trend towards the generation and production of increasing numbers of complex biopharmaceutical (protein based) products, there is an increased need and requirement to characterize both the product and production process in terms of robustness and reproducibility. This is of particular importance for products from mammalian cell culture which have large molecular structures and more often than not complex post-translational modifications (PTMs) that can impact the efficacy, stability and ultimately the safety of the final product. It is therefore vital to understand how the operating conditions of a bioprocess affect the distribution and make up of these PTMs to ensure a consistent quality and activity in the final product. Here we have characterized a typical bioprocess and determined (a) how the time of harvest from a mammalian cell culture and, (b) through the use of an ultra scale-down mimic how the nature of the primary recovery stages, affect the distribution and make up of the PTMs observed on a recombinant IgG(4) monoclonal antibody. In particular we describe the use of rapid whole antibody analysis by mass spectrometry to analyze simultaneously the changes that occur to the cleavage of heavy chain C-terminal lysine residues and the glycosylation pattern, as well as the presence of HL dimers. The time of harvest was found to have a large impact upon the range of glycosylation patterns observed, but not upon C-terminal lysine cleavage. The culture age had a profound impact on the ratio of different glycan moieties found on antibody molecules. The proportion of short glycans increased (e.g., (G0F)(2) 20-35%), with an associated decrease in the proportion of long glycans with culture age (e.g., (G2F)(2) 7-4%, and G1F/G2F from 15.2% to 7.8%). Ultra scale-down mimics showed that subsequent processing of these cultures did not change the post-translational modifications investigated, but did increase the proportion of half antibodies present in the process stream. The combination of ultra scale-down methodology and whole antibody analysis by mass spectrometry has demonstrated that the effects of processing on the detailed molecular structure of a monoclonal antibody can be rapidly determined early in the development process. In this study we have demonstrated this analysis to be applicable to critical process design decisions (e.g., time of harvest) in terms of achieving a desired molecular structure, but this approach could also be applied as a selection criterion as to the suitability of a platform process for the preparation of a new drug candidate. Also the methodology provides means for bioprocess engineers to predict at the discovery phase how a bioprocess will impact upon the quality of the final product., (2010 Wiley Periodicals, Inc.)

Regiospecific 3'-hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K(m) and k(cat) values of CYP105D7 for daidzein were 21.83 +/- 6.3 microM and 15.01 +/- 0.6 min(-1) in the presence of 1 microM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO-difference spectra at 450 nm using the whole cell extract. When the whole-cell reaction for the 3'-hydroxylation reaction of daidzein was carried out with 100 microM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6-fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3',4'-trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h., ((c) 2009 Wiley Periodicals.)

Post-translational limitations in the endoplasmic reticulum during recombinant monoclonal antibody production are an important factor in lowering the capacity for synthesis and secretion of correctly folded proteins. Mammalian protein disulfide isomerase (PDI) has previously been shown to have a role in the formation of disulfide bonds in immunoglobulins. Several attempts have been made to improve the rate of recombinant protein production by overexpressing PDI but the results from these studies have been inconclusive. Here we examine the effect of (a) transiently silencing PDI mRNA and (b) increasing the intracellular levels of members of the PDI family (PDI, ERp72, and PDIp) on the mRNA levels, assembly and secretion of an IgG4 isotype. Although transiently silencing PDI in NS0/2N2 cells suggests that PDI is involved in disulfide bond formation of this subclass of antibody, our results show that PDI does not control the overall IgG4 productivity. Furthermore, overexpression of members of the PDI family in a Chinese hamster ovary (CHO) cell line does not improve productivity and hence we conclude that the catalysis of disulfide bond formation is not rate limiting for IgG4 production., ((c) 2009 Wiley Periodicals, Inc.)

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species--Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression--evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)--correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production--evaluated as the ratio of the cell-specific production rate of GSH (q(GSH)) to the cell-specific production rate of THIOMAB (q(p))--corresponded to decreased 3LC levels. In time-lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and q(GSH)/q(p) ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high q(GSH)/q(p) ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity., ((c) 2009 Wiley Periodicals, Inc.)

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities., ((c) 2008 Wiley Periodicals, Inc.)

An innovative and automated method for the at-line monitoring of secreted protein was developed by harnessing a Surface Plasmon Resonance-based biosensor to a bioreactor. The proof of concept was performed by following at-line the relative concentration of a secreted protein produced by transient transfection of mammalian cells in a bioreactor. Our results suggest that our approach can be readily applied to the at-line determination of both protein concentration and bioactivity. Our experimental setup and strategy can thus satisfy the needs related to the development of novel bioprocess control protocols in the context of the new process analytical technology that arises in the biopharmaceutical industry., (Copyright 2007 Wiley Periodicals, Inc.)

The filamentous fungus, Aspergillus oryzae, was cultivated in batch and fed-batch cultivations in order to investigate the use of multi-wavelength fluorescence for monitoring course of events during filamentous fungi cultivations. The A. oryzae strain applied expressed a fungal lipase from Thermomyces lanuginosus. Spectra of multi-wavelength fluorescence were collected every 5 min with the BioView system (DELTA, Denmark) and both explorative and predictive models, correlating the fluorescence data with cell mass and lipase activity, were built. During the cultivations, A. oryzae displayed dispersed hyphal growth and under these conditions no fouling of the multi-wavelength fluorescence sensor was observed. The scores of a parallel factor analysis (PARAFAC) model, based on the fluorescence spectra, gave clear evidence of, for example, the on-set of the feeding phase. The predictive models, estimating the cell mass, showed correlations between 0.73 and 0.97 with root mean square error of cross validation (RMSECV) values between 1.48 and 0.77 g . kg(-1). A model estimating the lipase activity was also constructed for the fed-batch cultivations with a correlation of 0.93. The results presented here clearly show that multi-wavelength fluorescence is a useful tool for monitoring fed-batch cultivations of filamentous fungi., ((c) 2006 Wiley Periodicals, Inc.)

Glycosylation has profound effects on the quality of recombinant proteins produced in mammalian cells. The biosynthetic pathways of N-linked glycans on glycoproteins involves a relatively small number of enzymes and nucleotide sugars. Many of these glycoconjugate enzymes can utilize multiple N-glycans as substrates, thus generating a large number of glycan intermediates, and making the biosynthetic pathway resemble a network with diverging and converging paths. The N-glycans on secreted glycoprotein molecules include not only terminal glycans, but also pathway intermediates. To better assess the glycan distribution and the potential route of their synthesis, we created GlycoVis, a visualization program that displays the distribution and the potential reaction paths leading to each N-glycan on the reaction network. The substrate specificities of the enzymes involved were organized into a relationship matrix. With the input of glycan distribution data, the program outputs a reaction pathway map which labels the relative abundance levels of different glycans with different colors. The program also traces all possible reaction paths leading to each glycan and identifies each pathway on the map. Glycoform distribution of Chinese Hamster Ovary cell-derived tissue plasminogen activator (TPA), and human and mouse IgG were used as illustrations for the application of GlycoVis. In addition, the intracellular and secreted IgG from an NS0 producer cell line were isolated, and their glycoform profiles were displayed using GlycoVis for comparison. This visualization tool facilitates the analysis of potential reaction paths utilized under different physiological or culture conditions, and may provide insight on the potential targets for metabolic engineering., ((c) 2006 Wiley Periodicals, Inc.)

Cell encapsulation has been used to treat diabetes, amyotrophic lateral sclerosis, and other chronic ailments by the secretion of therapeutic proteins in vivo. Detection of these proteins typically requires invasive procedures such as blood sampling or device extraction, however. In this article, a non-invasive means of measuring secreted protein concentration using a co-expressed red fluorescent protein marker is developed. A bicistronic expression vector was constructed for the intracellular production of a red fluorescent protein marker and the secreted production of human interleukin-2 (hIL2). The destabilized red fluorescent protein, DsExDR, was selected for its rapid turnover, as well as its ability to emit red light, which is readily transmitted through mammalian tissue. Transfections of this bicistronic vector into three cell lines C2C12, HEK293, and Jurkat showed linear correlations between the expressed proteins, DsExDR (intracellular) and hIL2 (secreted), with transfection DNA concentration. Correspondingly, there was a linear correlation between secreted product (hIL2) and intracellular marker (DsExDR). As transfection DNA was increased, Jurkat cells were found to increase secreted hIL2 in direct proportion to the accumulated DsExDR. HEK293 and C2C12 cells expressed and secreted significantly more hIL2 than the Jurkat cells, while still maintaining a linear relationship. Thus, all three cell lines were suitable hosts for the bicistronic expression of DsExDR and expression and secretion of therapeutic hIL2. This reporting strategy may find the greatest use in cell encapsulation therapy., ((c) 2006 Wiley Periodicals, Inc.)

Microbial protein display technologies have enabled directed molecular evolution of binding and stability properties in numerous protein systems. In particular, dramatic improvements to antibody binding affinity and kinetics have been accomplished using these tools in recent years. Examples of successful application of display technologies to other immunological proteins have been limited to date. Herein, we describe the expression of human class II major histocompatibility complex allele (MHCII) HLA-DR4 on the surface of Saccharomyces cerevisiae as a noncovalently associated heterodimer. The yeast-displayed MHCII is fully native as assessed by binding of conformationally specific monoclonal antibodies; failure of antibodies specific for empty HLA-DR4 to bind yeast-displayed protein indicates antigenic peptide is bound. This report represents the first example of a noncovalent protein dimer displayed on yeast and of successful display of wild-type MHCII. Results further point to the potential for using yeast surface display for engineering and analyzing the antigen binding properties of MHCII.