Gkoutzioupa, Victoria; (2024) Integrated Experimental Investigation of Upstream and Primary Recovery Operations to Improve Mammalian Cell Based Process Performance. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text Gkoutzioupa_Thesis.pdf - Other Download (10MB) | Preview

Abstract

Monoclonal antibodies (mAb) are therapeutic proteins that are widely used for the treatment of a variety of diseases, including cancer and autoimmune disorders. The antibody market is constantly increasing, with 162 therapies being approved worldwide by 30 June 2022 (Lyu et al., 2022). The expiration of the original mAb patents and the introduction of biosimilars and biobetters in the market, increases the need for rapid and cost-efficient manufacturing. The traditional approach where only one unit operation at a time is optimised, creates a knowledge gap between upstream and downstream processing, leading to inefficient, time-consuming and expensive solutions. In this thesis a holistic approach was followed with the aims of increasing the final titer, decreasing the host cell proteins (HCP) at harvest and improving the depth filtration performance in terms harvest cell culture fluid (HCCF) processed. Initially, scale-down studies were conducted upstream, from 5 L STR to 250 mL STR and 1 L shake flasks, and in primary recovery from 270 cm2 to 23 cm2 depth filters, using a producing GS-CHO cell line. The reason for the scale down studies was to investigate the effect of scaling, controlled versus uncontrolled operating conditions and different agitation systems on process performance, upstream and during primary recovery. Additionally, most of the experiments of the thesis were conducted in 250 mL and 1 L shake flasks, hence, it was important to investigate how the findings related to benchtop scale bioreactors. The scale down models had comparable growth curves and titer production, however they were different in depth filtration performance, with the 5 L STR performing significantly better. This enhanced behaviour could be mainly attributed to the low flow sensor accuracy in small scales leading to increased flow rates. A 3-level full factorial DoE methodology, carried out in 1 L shake flasks, was used to explore the effects and interactions of critical process parameters (CPP), such as osmolality and temperature shifts on mAb titre, HCP production and HCCF filtrate volumes during depth filtration. The three factors were: (a) temperature shift (32oC, 34.5oC, and 37oC), (b) osmolality shift (320 mOsm∙kg-1, 380 mOsm∙kg-1, 440 mOsm∙kg-1) and (c) cell line (GS-CHO IgG4 producing, GS-CHO non-producing, CHO-S trastuzumab producing). The shifts from control conditions (T=37oC, Osmo=320 mOsm∙kg-1) took place in the end of exponential phase and gave different responses in the three cell lines. In GS-CHO cells, shift to 32oC prolonged the culture duration by 10 days and increased the titer 1.23 times compared to control conditions, while shift to 440 mOsm∙kg-1, prolonged the depth filtration duration by 22 minutes. Similar findings to producing GS-CHO cells, were reported for the null GS-CHO cells. In contrast, CPP shifts affected negatively the filterability of the CHO-S cell HCCF. Overall, among the cell lines, taking into account all the conditions, CHO-S cells had the worst performance, since they produced less titer, more HCP and processed less HCCF volume. A one-factor-a-time (OFAT) investigation was performed in 250 mL STR using the GS-CHO producing cell line. The three factors that were considered were: (a) pH shift (6.8, 7.2, and 7.5), (b) temperature shift (32oC, 34.5oC, and 37oC) and (c) osmolality shift (380 mOsm∙kg-1, 450 mOsm∙kg-1 and 520 mOsm∙kg-1. Similarly to shake flask experiments, the highest titer was achieved in cultures that shifted to 32oC and it was 1.15 times higher than control conditions, while the depth filtration process was prolonged by 13 minutes when shifting to 520 mOsm∙kg-1. In 5 L STR, a temperature shift to 32oC, resulted in a 1.25 times titer increase. In summary, the study of GS-CHO producing cell cultures in different scales (1 L shake flasks, 250 mL STR, 5 L STR) showed that 1 L shake flasks could be used for optimisation studies since, although being uncontrolled systems, with different agitation mechanisms, they could still provide comparable results. The DoE experiments suggested that the 3 cell lines had significant differences in terms of their responses in temperature and osmolality changes. Regarding GS-CHO cells, DoE outcomes showed that hypothermic conditions could increase mAb titer production, while hyperosmotic could improve the depth filtration performance in terms of HCCF volume filtered and HCP removal. These finding were confirmed in 250 mL and 5 L bioreactor scale.

Download activity - last month

Export as CSVJSONXML

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as XMLJSONCSV

Archive Staff Only

View Item