Cascading effect in bioprocessing—The impact of mild hypothermia on CHO cell behavior and host cell protein composition

Abstract

A major challenge in downstream purification of monoclonal antibodies (mAb) is the removal of host cell proteins (HCPs). Previous studies have shown that cell culture conditions significantly impact the HCP content at harvest. However, it is currently unclear how process conditions affect physiological changes in the host cell population, and how these changes, in turn, cascade down to change the HCP profile. We examined how temperature downshift (TDS) to mild hypothermia affects key upstream performance indicators, that is antibody titre, HCP concentration and HCP species, across the cell culture decline phase and at harvest through the lens of changes in cellular behavior. Mild hypothermic conditions introduced on day 5 of fed-batch Chinese hamster ovary (CHO) cell bioreactors resulted in a lower cell proliferation rate but larger percentages of healthier cells across the cell culture decline phase compared to bioreactors maintained at standard physiological temperature. Moreover, the onset of apoptosis was less evident in mild hypothermic cultures. Consequently, mild hypothermic cultures took an extra 5 days to reach an integral viable cell concentration (IVCC) and antibody yield similar to that of the control at standard physiological temperature. When cell viability dropped below 80%, mild hypothermic cell cultures had a reduced variety of HCP species by 36%, including approximately 44% and 27% lower proteases and chaperones, respectively, despite having similar HCP concentration. This study suggests that TDS may be a good strategy to provide cleaner downstream feedstocks by reducing the variety of HCPs and to maintain product integrity by reducing the number of proteases and chaperones.

CHO cell cultures in shake flasks and bioreactors present different host cell protein profiles in the supernatant

“…Interestingly, cell viability was maintained at high percentages in mild hypothermic bioreactors operated in similar working volume, stirring speed and aeration rate as the control. Cells at 32C appeared to be more robust than the control which is believed to be a result of homeoviscous adaptation of the cell membrane at lower culture temperature [12,13,27].…”

“…Furthermore, HCPs such as chaperones and proteases may compromise product quality by promoting product aggregation or fragmentation [8][9][10]. In the past five years, the concept of Quality by Design (QbD) had been employed to understand the impact of upstream culture conditions like cell age, cell culture mode (batch or fed-batch), process duration and culture temperature on HCP profile [11][12][13][14] with an aim to reduce the level of HCPs in downstream feedstock by optimizing cell culture conditions.…”

“…Several of these studies were carried out in flasks [4,11], while others performed research with bioreactors that mimic typical biopharmaceutical productions [13][14][15]. However, our understanding of how different culture methods in flasks versus bioreactors affect the extracellular HCP profile at harvest is limited.…”

“…For example, does scale-up from flasks to bioreactors change the dynamics between cellular behavior and the corresponding HCP profile? We recently reported that the HCP level in supernatant collected from bioreactors increases proportionally with the percentage of dead cells, while the degree of apoptosis determines the variety of HCP species found in the supernatant [13]. Would these correlations have been identified had the study been conducted in shake flasks?…”

Understanding the mechanism of copurification of “difficult to remove” host cell proteins in rituximab biosimilar products

“…With the rise of biosimilars across the globe, process changes are inevitable as biosimilar manufacturers do not have access to the process used by the manufacturer of the innovator product . Researchers have shown that these process changes or manufacturing drifts could influence both the levels of HCPs as well the HCP species finally end up in the biotherapeutic product . Thus, while the criterion of 100 ppm target limit may be satisfied, manufacturers need a deeper understanding of the HCPs present in the product and their impact on the product's safety and efficacy.…”