Production of butyrate and branched-chain amino acid catabolic byproducts by CHO cells in fed-batch culture enhances their specific productivity.

Chinese hamster ovary (CHO) cells in fed-batch cultures produce several metabolic byproducts derived from amino acid catabolism, some of which accumulate to growth inhibitory levels. Controlling the accumulation of these byproducts has been shown to significantly enhance cell proliferation. Interestingly, some of these byproducts have physiological roles that go beyond inhibition of cell proliferation. In this study, we show that, in CHO cell fed-batch cultures, branched-chain amino acid (BCAA) catabolism contributes to the formation of butyrate, a novel byproduct that is also a well-established specific productivity enhancer. We further show that other byproducts of BCAA catabolism, namely isovalerate and isobutyrate, which accumulate in CHO cell fed-batch cultures, also enhance specific productivity. Lastly, we show that the rate of production of these BCAA catabolic byproducts is negatively correlated with glucose uptake and lactate production rates. Thus, limiting glucose supply to suppress glucose uptake and lactate production, as in the case of fed-batch cultures employing high-end pH-controlled delivery of glucose (HiPDOG) technology, significantly enhances BCAA catabolic byproduct accumulation, resulting in higher specific productivities.

Hydrocyclones as cell retention devices for an N-1 perfusion bioreactor linked to a continuous-flow stirred tank production bioreactor.

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.

Novel, linked bioreactor system for continuous production of biologics.

A novel, alternative intensified cell culture process comprised of a linked bioreactor system is presented. An N-1 perfusion bioreactor maintained cells in a highly proliferative state and provided a continuous inoculum source to a second bioreactor operating as a continuous-flow stirred-tank reactor (CSTR). An initial study evaluated multiple system steady-states by varying N-1 steady-state viable cell densities, N-1 to CSTR working volume ratios, and CSTR dilution rates. After identifying near optimum system steady-state parameters yielding a relatively high volumetric productivity while efficiently consuming media, a subsequent lab-scale experiment demonstrated the startup and long-term operation of the envisioned manufacturing process for 83 days. Additionally, to compensate for the cell-specific productivity loss over time due to cell line instability, the N-1 culture was also replaced with younger generation cells, without disturbing the steady-state of the system. Using the model cell line, the system demonstrated a two-fold volumetric productivity increase over the commercial-ready, optimized fed-batch process.

Metabolic engineering of Chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures.

Chinese hamster ovary (CHO) cells in fed-batch cultures are known to consume large amounts of nutrients and divert significant portion of them towards the formation of byproducts, some of which, including lactate and ammonia, are known to be growth inhibitory in nature. A major fraction of these inhibitory metabolites are byproducts or intermediates of amino acid catabolism. Limiting the supply of amino acids has been shown to curtail the production of corresponding inhibitory byproducts resulting in enhanced growth and productivities in CHO cell fed-batch cultures (Mulukutla et al., 2017). In the current study, metabolic engineering of CHO cells was undertaken in order to reduce the biosynthesis of these novel growth inhibitors. Phenylalanine-tyrosine (Phe-Tyr) and branched chain amino acid (BCAA) catabolic pathways were engineered as part of this effort. Four genes that encode enzymes in the Phe-Tyr pathway, which were observed to be minimally expressed in CHO cells, were in turn overexpressed. Metabolically engineered cells were prototrophic to tyrosine and had reduced production of the inhibitory byproducts from Phe-Tyr pathway including 3-phenyllactate and 4-hydroxyphenyllactate. In case of BCAA catabolic pathway, branched chain aminotransferase 1 (BCAT1) gene, which encodes the enzyme that catalyzes the first step in the catabolism of BCAAs, was knocked out in CHO cells. Knockout (KO) of BCAT1 function completely eliminated production of inhibitory byproducts from BCAA catabolic pathway, including isovalerate, isobutyrate and 2-methylbutyrate, resulting in significantly enhanced cell growth and productivities in fed-batch cultures. This study is first of its kind to demonstrate that metabolic engineering of essential amino acid metabolism of CHO cells can significantly improve cell culture process performance.

Shift to high-intensity, low-volume perfusion cell culture enabling a continuous, integrated bioprocess.

In order to address the increasing demand for biologics, cell culture intensification using perfusion offers significantly higher productivities while also reducing manufacturing costs, especially when part of an integrated, continuous bioprocess. An initial study of a long-duration perfusion process using a cell-bleed to maintain a target cell density observed a 2.1-fold higher cell-specific productivity and a gradual decline in the culture growth rate when perfused at an overall lower rate. Subsequent studies sought an alternative process that largely reduced the overall volume of media needed by first perfusing at a high cell-specific perfusion rate (CSPR) to support a high cell density followed by continued perfusion at a low CSPR to promote a more productive stationary phase. This high intensity, low-volume perfusion (HILVOP) process achieved cumulative volumetric productivities of 1.5-1.6 g/L/day with two CHO cell lines. When compared to each cell line's respective commercial-ready, fed-batch process, a 3.1-3.8-fold productivity increase was demonstrated while yielding similar product quality. Furthermore, the higher productivity achieved with HILVOP used 6.6-12.3-fold less media than a similarly productive long-duration process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1472-1481, 2018.

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. © 2017 Wiley Periodicals, Inc.

Cell-controlled hybrid perfusion fed-batch CHO cell process provides significant productivity improvement over conventional fed-batch cultures.

A simple method originally designed to control lactate accumulation in fed-batch cultures of Chinese Hamster Ovary (CHO) cells has been modified and extended to allow cells in culture to control their own rate of perfusion to precisely deliver nutritional requirements. The method allows for very fast expansion of cells to high density while using a minimal volume of concentrated perfusion medium. When the short-duration cell-controlled perfusion is performed in the production bioreactor and is immediately followed by a conventional fed-batch culture using highly concentrated feeds, the overall productivity of the culture is approximately doubled when compared with a highly optimized state-of-the-art fed-batch process. The technology was applied with near uniform success to five CHO cell processes producing five different humanized monoclonal antibodies. The increases in productivity were due to the increases in sustained viable cell densities. Biotechnol. Bioeng. 2017;114: 1438-1447. © 2017 Wiley Periodicals, Inc.