Bhanu Chandra Mulukutla

On metabolic shift to lactate consumption in fed-batch culture of mammalian cells.

Fedbatch culture is the prevalent cell cultivation method in producing protein therapeutics. A metabolic shift to lactate consumption in late stage of cultivation has been shown to extend the culture viability and increase product concentrations. To better understand the factors, which trigger metabolic shift we performed transcriptome and metabolic flux analysis on a fedbatch culture of mouse myeloma cell line (NS0) and developed a mechanistic kinetic model for energy metabolism. Experimental observation indicates that the shift to lactate consumption occurs upon the cessation of rapid growth and under conditions of low glycolysis flux and high extracellular lactate concentrations. Although the transition is accompanied by a general down regulation of enzymes in energy metabolism, that alone was insufficient to elicit a metabolic shift. High lactate level has been reported to exert an inhibitory effect on glycolysis enzyme phosphofructokinase; model simulation suggests that a high lactate level can contribute to reduced glycolytic flux as well as providing a driving force for its conversion to pyruvate. The transcriptome data indicate that moderate alteration in the transcript levels of AKT1 and P53 signaling pathways genes occurs in the late stage of culture. These signaling pathways are known to regulate glycolytic activity. Model simulations further suggest that AKT1 signaling plays a key role in facilitating lactate consumption. Collectively, our results strongly suggest that lactate consumption in fedbatch culture is an outcome of reduced glycolysis flux, which is a product of lactate inhibition and regulatory action of signaling pathway caused by reduced growth rate.

Systems Analysis of N-Glycan Processing in Mammalian Cells

N-glycosylation plays a key role in the quality of many therapeutic glycoprotein biologics. The biosynthesis reactions of these oligosaccharides are a type of network in which a relatively small number of enzymes give rise to a large number of N-glycans as the reaction intermediates and terminal products. Multiple glycans appear on the glycoprotein molecules and give rise to a heterogeneous product. Controlling the glycan distribution is critical to the quality control of the product. Understanding N-glycan biosynthesis and the etiology of microheterogeneity would provide physiological insights, and facilitate cellular engineering to enhance glycoprotein quality. We developed a mathematical model of glycan biosynthesis in the Golgi and analyzed the various reaction variables on the resulting glycan distribution. The Golgi model was modeled as four compartments in series. The mechanism of protein transport across the Golgi is still controversial. From the viewpoint of their holding time distribution characteristics, the two main hypothesized mechanisms, vesicular transport and Golgi maturation models, resemble four continuous mixing-tanks (4CSTR) and four plug-flow reactors (4PFR) in series, respectively. The two hypotheses were modeled accordingly and compared. The intrinsic reaction kinetics were first evaluated using a batch (or single PFR) reactor. A sufficient holding time is needed to produce terminally-processed glycans. Altering enzyme concentrations has a complex effect on the final glycan distribution, as the changes often affect many reaction steps in the network. Comparison of the glycan profiles predicted by the 4CSTR and 4PFR models points to the 4PFR system as more likely to be the true mechanism. To assess whether glycan heterogeneity can be eliminated in the biosynthesis of biotherapeutics the 4PFR model was further used to assess whether a homogeneous glycan profile can be created through metabolic engineering. We demonstrate by the spatial localization of enzymes to specific compartments all terminally processed N-glycans can be synthesized as homogeneous products with a sufficient holding time in the Golgi compartments. The model developed may serve as a guide to future engineering of glycoproteins.

Glucose metabolism in mammalian cell culture: new insights for tweaking vintage pathways

Cultured mammalian cells are major vehicles for producing therapeutic proteins, and energy metabolism in those cells profoundly affects process productivity. The characteristic high glucose consumption and lactate production of industrial cell lines as well as their adverse effects on productivity have been the target of both cell line and process improvement for several decades. Recent research advances have shed new light on regulation of glucose metabolism and its links to cell proliferation. This review highlights our current understanding in this area of crucial importance in bioprocessing and further discusses strategies for harnessing new findings toward process enhancement through the manipulation of cellular energy metabolism.

Developing genomic platforms for Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used in recombinant protein production, yet despite their importance in bioprocessing, few genomic resources have been developed for this cell line. Over the past several years, we have made considerable progress in the development of genomic tools for CHO. Using Sanger-based sequencing technology, we have accrued a sequence repertoire of more than 68,000 expressed sequence tags (ESTs), representing more than 28,000 unique CHO transcripts. Using closely related species, we have functionally annotated this sequence set and have currently achieved significant representation in a number of functional classes, including some closely tied to recombinant protein production. This sequence repository has been used to design custom CHO Affymetrix arrays for transcriptome analysis. Illumina Solexa deep sequencing technology was also applied to study the CHO cell transcriptome and survey the identity and expression of small RNAs. These applications demonstrate the utility of genomic tools, and illustrate the applicability of emerging next-generation sequencing technologies.

Reaching the depth of the Chinese hamster ovary cell transcriptome

The high-throughput DNA sequencing Illumina Solexa GAII platform was employed to characterize the transcriptome of an antibody-producing Chinese hamster ovary (CHO) cell line. More than 55 million sequencing reads were generated and mapped to an existing set of CHO unigenes derived from expressed sequence tags (ESTs), as well as several public sequence databases. A very significant fraction of sequencing reads has not been previously seen. The frequency with which fragments of a unigene were sequenced was taken as an estimate of the abundance level of the corresponding transcripts. A wide dynamic range of transcript abundance levels was observed, spanning six orders of magnitude. However, the distribution of coverage across transcript lengths was found to vary, from relatively uniform to highly variable. This observation suggests that more challenges are yet to be resolved before direct sequencing can be used as a true quantitative measure of transcript level and for differential gene expression analysis. With the depth that high-throughput sequencing methods can reach, one can expect that the entire transcriptome of this industrially important organism will be decoded in the near future.

Bistability in glycolysis pathway as a physiological switch in energy metabolism.

The flux of glycolysis is tightly controlled by feed-back and feed-forward allosteric regulations to maintain the bodys glucose homeostasis and to respond to cells growth and energetic needs. Using a mathematical model based on reported mechanisms for the allosteric regulations of the enzymes, we demonstrate that glycolysis exhibits multiple steady state behavior segregating glucose metabolism into high flux and low flux states. Two regulatory loops centering on phosphofructokinase and on pyruvate kinase each gives rise to the bistable behavior, and together impose more complex flux control. Steady state multiplicity endows glycolysis with a robust switch to transit between the two flux states. Under physiological glucose concentrations the glycolysis flux does not move between the states easily without an external stimulus such as hormonal, signaling or oncogenic cues. Distinct combination of isozymes in glycolysis gives different cell types the versatility in their response to different biosynthetic and energetic needs. Insights from the switch behavior of glycolysis may reveal new means of metabolic intervention in the treatment of cancer and other metabolic disorders through suppression of glycolysis.

Multiplicity of Steady States in Glycolysis and Shift of Metabolic State in Cultured Mammalian Cells

Cultured mammalian cells exhibit elevated glycolysis flux and high lactate production. In the industrial bioprocesses for biotherapeutic protein production, glucose is supplemented to the culture medium to sustain continued cell growth resulting in the accumulation of lactate to high levels. In such fed-batch cultures, sometimes a metabolic shift from a state of high glycolysis flux and high lactate production to a state of low glycolysis flux and low lactate production or even lactate consumption is observed. While in other cases with very similar culture conditions, the same cell line and medium, cells continue to produce lactate. A metabolic shift to lactate consumption has been correlated to the productivity of the process. Cultures that exhibited the metabolic shift to lactate consumption had higher titers than those which didn’t. However, the cues that trigger the metabolic shift to lactate consumption state (or low lactate production state) are yet to be identified. Metabolic control of cells is tightly linked to growth control through signaling pathways such as the AKT pathway. We have previously shown that the glycolysis of proliferating cells can exhibit bistability with well-segregated high flux and low flux states. Low lactate production (or lactate consumption) is possible only at a low glycolysis flux state. In this study, we use mathematical modeling to demonstrate that lactate inhibition together with AKT regulation on glycolysis enzymes can profoundly influence the bistable behavior, resulting in a complex steady-state topology. The transition from the high flux state to the low flux state can only occur in certain regions of the steady state topology, and therefore the metabolic fate of the cells depends on their metabolic trajectory encountering the region that allows such a metabolic state switch. Insights from such switch behavior present us with new means to control the metabolism of mammalian cells in fed-batch cultures.

Regulation of Glucose Metabolism – A Perspective From Cell Bioprocessing

Cultured mammalian cells are the main workhorses for producing biologics. The performance of these cell culture processes, in terms of both productivity and product quality attributes, is significantly influenced by cellular metabolism. Glucose is the major carbon source for cellular biosynthesis and energy generation. We summarize here recent advances in our understanding of the regulation of glucose metabolism in cultured cells. The versatility of cells to sustain homeostatic states under widely varying environments is made possible by allosteric regulation of the metabolic network, interplay between the signaling pathways, and transcription factors. Understanding the regulation of metabolism holds the key to altering the metabolic regulatory circuit and implementing direct metabolic control over cell culture processes.

Identification and control of novel growth inhibitors in fed-batch cultures of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells in culture are known to consume large amounts of nutrients and divert most of them toward byproducts, some of which, including lactate and ammonia, are known to be toxic in nature. Glucose limitation strategies can successfully control lactate accumulation in fed‐batch cultures yielding higher peak cell densities and titers. Interestingly, even in such optimized cultures, cell growth slows and eventually stops, indicating the emergence of other factors that negatively affect cell growth. In this study, we employed omics techniques to identify and quantify nine compounds that are intermediates or byproducts of amino acid metabolism, and accumulate in fed‐batch cultures. Treatment with these compounds either individually or in a combined fashion resulted in partial or complete cell growth inhibition. Careful control of selected amino acid concentrations between one‐half and one millimolar during the growth phase of fed‐batch cultures reduced accumulation of the inhibitory metabolites and allowed for higher peak cell densities and increased productivity. Biotechnol. Bioeng. 2017;114: 1779–1790.

Mechanism for Multiplicity of Steady States with Distinct Cell Concentration in Continuous Culture of Mammalian Cells

Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi‐scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells. Biotechnol. Bioeng. 2015;112: 1437–1445.