Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures.

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d-glucose (2DG) and 5-thio- d-glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%-45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to 23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%-33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.

Perfusion Cell Culture Decreases Process and Product Heterogeneity in a Head-to-Head Comparison With Fed-Batch.

In this study, the authors compared the impacts of fed-batch and perfusion platforms on process and product attributes for IgG1- and IgG4-producing cell lines. A "plug-and-play" approach is applied to both platforms at bench scale, using commercially available basal and feed media, a standard feed strategy for fed-batch and ATF filtration for perfusion. Product concentration in fed-batch is 2.5 times greater than perfusion, while average productivity in perfusion is 7.5 times greater than fed-batch. PCA reveals more variability in the cell environment and metabolism during the fed-batch run. LDH measurements show that exposure of product to cell lysate is 7-10 times greater in fed-batch. Product analysis shows larger abundances of neutral species in perfusion, likely due to decreased bioreactor residence times and extracellular exposure. The IgG1 perfusion product also has higher purity and lower half-antibody. Glycosylation is similar across both culture modes. The first perfusion harvest slice for both product types shows different glycosylation than subsequent harvests, suggesting that product quality lags behind metabolism. In conclusion, process and product data indicate that intra-lot heterogeneity is decreased in perfusion cultures. Additional data and discussion is required to understand the developmental, clinical and commercial implications, and in what situations increased uniformity would be beneficial.

Integrated continuous production of recombinant therapeutic proteins.

In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.

Production, characterization, and pharmacokinetic properties of antibodies with N-linked mannose-5 glycans.

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.

Effects of cell culture conditions on antibody N-linked glycosylation--what affects high mannose 5 glycoform.

The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process.

Understanding the intracellular effect of enhanced nutrient feeding toward high titer antibody production process.

One of the major goals in cell culture process development for therapeutic antibody production is to develop methods to reach high titer in classical fed-batch processes. This goal is often achieved through the optimizations of expression vector, cell line, media and cell culture process controls to increase cell specific productivity, viable cell density, and culture longevity. During process optimization for a selected production cell line, cell specific productivity (qP) can vary significantly with culture conditions. Therefore, identifying strategies to maintain maximal specific productivity throughout the entire fed-batch culture and to eliminate cellular/process bottlenecks that prevent high levels of antibody production would be crucial for further advancements in this area. In this work, specific productivity was increased and maintained at high level throughout the course of the culture by the optimization of feed media and feeding strategy. Through the enhancement of nutrient feeding, final titer was increased by 2.5-fold from the platform fed-batch process and reached 7.5 g/L. In addition, further insight upon possible cellular bottlenecks in high yield antibody production was obtained by comparing the levels of heavy chain (HC) and light chain (LC) mRNA and the levels of intracellular antibody between the non-optimized and optimized feeding processes. The mRNA levels of the two processes were measured and exhibited no significant difference suggesting that transcription is not the bottleneck. When intracellular antibody level was studied, the relatively constant level of HC, LC, and intact antibody between days 9 and 14 suggested that translation could be the rate-limiting step under the non-optimized nutrient feeding condition due to the dramatic drop of qP to roughly zero which correlated with the depletion of tyrosine as one of the key amino acids for protein synthesis. Finally, accumulation of unassembled HC but not intact antibody was observed at days 14-18 under the enhanced feeding condition, implying that folding and assembly may be the bottleneck toward the end of the culture.

Characterization of a key trifunctional enzyme for aromatic amino acid biosynthesis in Archaeoglobus fulgidus.

In the aromatic amino acid biosynthesis pathway, chorismate presents a branch point intermediate that is converted to tryptophan, phenylalanine (Phe), and tyrosine (Tyr). In bacteria, three enzymes catalyze the conversion of chorismate to hydroxyphenylpyruvate or pyruvate. The enzymes, chorismate mutase (CM), prephenate dehydratase (PDT), and prephenate dehydrogenase (PDHG) are either present as distinct proteins or fusions combining two activities. Gene locus AF0227 of Archaeoglobus fulgidus is predicted to encode a fusion protein, AroQ, containing all three enzymatic domains. This work describes the first characterization of a trifunctional AroQ. The A. fulgidus aroQ gene was cloned and overexpressed in Escherichia coli. The recombinant protein purified as a homohexamer with specific activities of 10, 0.51, and 50 U/mg for CM, PDT, and PDHG, respectively. Tyr at 0.5 mM concentration inhibited PDHG activity by 50%, while at 1 mM PDT was activated by 70%. Phe at 5 muM inhibited PDT activity by 66% without affecting the activity of PDHG. A fusion of CM, PDT, and PDHG domains is evident in the genome of only one other organism sequenced to date, that of the hyperthermophilic archaeon, Nanoarchaeum equitans. Such fusions of contiguous activities in a biosynthetic pathway may constitute a primitive strategy for the efficient processing of labile metabolites.

Encapsulation of semiconducting polymers in vault protein cages.

We demonstrate that a semiconducting polymer [poly(2-methoxy-5-propyloxy sulfonate phenylene vinylene), MPS-PPV] can be encapsulated inside recombinant, self-assembling protein nanocapsules called "vaults". Polymer incorporation into these nanosized protein cages, found naturally at approximately 10,000 copies per human cell, was confirmed by fluorescence spectroscopy and small-angle X-ray scattering. Although vault cellular functions and gating mechanisms remain unknown, their large internal volume and natural prevalence within the human body suggests they could be used as carriers for therapeutics and medical imaging reagents. This study provides the groundwork for the use of vaults in encapsulation and delivery applications.

Reversible pH lability of cross-linked vault nanocapsules.

Vaults are ubiquitous, self-assembled protein nanocapsules with dimension in the sub-100 nm range that are conserved across diverse phyla from worms to humans. Their normal presence in humans at a copy number of over 10,000/cell makes them attractive as potential drug delivery vehicles. Toward this goal, bifunctional amine-reactive reagents are shown to be useful for the reversible cross-linking of recombinant vaults such that they may be closed and opened in a controllable manner.

Case study and application of process analytical technology (PAT) towards bioprocessing: use of on-line high-performance liquid chromatography (HPLC) for making real-time pooling decisions for process chromatography.

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. This paper presents a PAT application for one of the most commonly used unit operation in bioprocessing, namely liquid chromatography. Feasibility of using a commercially available online-high performance liquid chromatography (HPLC) system for real-time pooling of process chromatography column is examined. Further, experimental data from the feasibility studies are modeled and predictions of the model are compared to actual experimental data. It is found that indeed for the application under consideration, the online-HPLC offers a feasible approach for analysis that can facilitate real-time decisions for column pooling based on product quality attributes. It is shown that implementing this analytical scheme allows us to meet two of the key goals that have been outlined for PAT, that is, "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." Finally, the implications of implementing such a PAT application in a manufacturing environment are discussed. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis.

Vault nanocapsule dissociation into halves triggered at low pH.

Vaults are self-assembled ribonucleoprotein nanocapsules that consist of multiple copies of three proteins (major vault protein, VPARP, and TEP1) and an untranslated RNA. Although their function has not been determined, vaults are found in nearly all eukaryotic cells. This study describes the use of fluorescence spectroscopy, multiangle laser light scattering (MALLS), and the quartz crystal microbalance (QCM) as tools in investigating recombinant vault conformational change in response to a varied solution pH. Identification of conditions for reversible vault disassembly and reassembly could enable application of these nanocapsules in drug delivery and in nanomaterials synthesis. Initial monitoring of changes in the intrinsic fluorescence intensity of vaults showed a 60% increase at pH 3.4 compared to that at pH 6.5, suggesting vaults exhibit a more open conformation at low pH. Fluorescence quenching studies provided further evidence of a vault structural change at low pH. MALLS data suggested a decrease in molecular mass accompanied by a clear increase in the radius of gyration as the solution pH was shifted from 6.5 to 3.4. This result prompted the hypothesis that vaults dissociate at least partially at low pH. Using the QCM to study adsorption of the vault onto self-assembled monolayers, data that suggest vault dissociation at low pH, even when the vault is in an adsorbed state, were also obtained. Finally, transmission electron microscopy (TEM) of negatively stained vaults at pH 6.5 and 3.4 confirmed the fluorescence spectroscopy, MALLS, and QCM findings by providing visual evidence that vaults disassemble into halves as the solution pH is lowered from 6.5 to 3.4.