Gregory Walter Hiller

High-End pH-Controlled Delivery of Glucose Effectively Suppresses Lactate Accumulation in CHO Fed-Batch Cultures

A simple method for control of lactate accumulation in suspension cultures of Chinese hamster ovary (CHO) cells based on the cultures pH was developed. When glucose levels in culture reach a low level (generally below 1 mM) cells begin to take up lactic acid from the culture medium resulting in a rise in pH. A nutrient feeding method has been optimized which delivers a concentrated glucose solution triggered by rising pH. We have shown that this high‐end pH‐controlled delivery of glucose can dramatically reduce or eliminate the accumulation of lactate during the growth phase of a fed‐batch CHO cell culture at both bench scale and large scale (2,500 L). This method has proven applicable to the majority of CHO cell lines producing monoclonal antibodies and other therapeutic proteins. Using this technology to enhance a 12‐day fed‐batch process that already incorporated very high initial cell densities and highly concentrated medium and feeds resulted in an approximate doubling of the final titers for eight cell lines. The increase in titer was due to additional cell growth and higher cell specific productivity. Biotechnol. Bioeng. 2011; 108:1328–1337.

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.

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.

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 lines 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.

A High Cell Density Approach to Fed-Batch Cell Culture for Production of Biopharmaceuticals

We have evaluated a more cell-intensive approach to both cell line adaptation and production culture as a means of achieving high integrated viable cell density (IVCD) in order to improve cell culture process yields. Shake flask based Fed-batch (FB) production cultures were seeded at multiple cell densities. Increases in seed density in the fed batch up to 8x control seed density resulted in significantly higher IVCD and titer with a 2–3 fold benefit relative to control seeded cultures. Whereas the initial increments in production seed density resulted in a linear increase in IVCD and titer, further increments showed diminishing returns. It appeared that the highest seed density production cultures were limited by nutrient supply and so richer medium and a higher feeding rate were then tested, resulting in further improvements in IVCD and titer. We also tested the effect of adapting the cells to high densities for over 50 generations prior to production cultures. For the production assessment, both the high- and standard-density adapted cells were seeded at multiple cell densities. Effect of the adaptation was modest and variable. By combining high-seed adaptation, high production culture seed densities, enriched media and high feeding rate titer yields of up to 9 g/L were achieved.