Identification of upstream culture conditions and harvest time parameters that affect host cell protein clearance

Abstract

During early stage bioprocess development, characterizing interactions between unit operations is a key challenge. Such interactions include the release of host cell enzymes early in the process causing losses in product quality downstream.Using a CHO-expressed IgG1 system, the impact of cell culture duration was investigated using a 50 L bioreactor and performing scale-down protein A purification. While antibody titer doubled during the last week of culture, the post-protein A host cell protein (HCP) levels increased from 243 to 740 ppm. Effects of pH and temperature were then explored using fed-batch ambr250 bioreactors, and parameters enabling higher titers were linked to a decrease in post-protein A product purity. These trade-offs between titer and product quality were visualized using a window of operation. The downstream space was explored further by exposing shake flask material to shear representative of disc stack centrifugation, prior to purification, and by adding polishing chromatography. While product quality decreased with progressing cultivation, cells became more shear resistant. Polishing chromatography resulted in product fragmentation which increased fourfold from Day 10 to 24, adding constraint to achieving both efficient HCP clearance as well as high monomer purities. These examples highlight the importance of adopting integrated approaches to upstream and downstream development strategies to enable whole process optimization. K E Y W O R D S downstream processing, host cell proteins, monoclonal antibodies, product quality, upstream processing

Performance of an acoustic settler versus a hollow fiber–based ATF technology for influenza virus production in perfusion

“…To better assess the performance of a process, upstream and downstream aspects should be considered together (Agarabi et al 2017;Wilson et al 2019). Results showed similar host cell dsDNA and total protein contamination levels, fewer large-sized virus and other aggregates, and significant increase in volumetric virus productivity, when using an acoustic settler in comparison with an ATF system.…”

Monitoring Parallel Robotic Cultivations with Online Multivariate Analysis

“…Here we present a program to monitor parallel bioreactor systems in high-throughput microbial cultivations. With this program, we can supervise if replicates follow a coherent pattern throughout (16,17,18; feed rate: µ set = 40% of µ max ). The DOT profiles of Reactor 16 deviates from its group of replicates, indicating the system stops reacting to the pulse-based feeding.…”

“…The technical requirements are already met by modern HT robots with developments at the µL scale. Significant advantages are offered by mini-bioreactors (MBR) with working volumes between 10 and 250 mL [17][18][19], integrated online sensors for pH and DOT, integrated control of pH and substrate feeding [20][21][22] and automated at-line sampling and analysis [8,21,23]. Additionally, computer aided tools that enable advanced process control and feedback operation of the robots and the MBRs have been developed, but are rarely used [17,18,20,22,[24][25][26].…”

“…Aeration system blockage. (a) Trajectories of the DOT profile from 07:50-08:50 h into the cultivation with highlighted reactors(16,17,18; feed rate: μset = 40% of μmax).The DOT profiles of Reactor 16 deviates from its group of replicates, indicating the system stops reacting to the pulsebased feeding. (b,c) Score plot based on the sliding window PCA model with input variables pH, DOT, base volume addition, and feed volume addition in the time range t0 = 08:10 h to tend = 08:30 h (b) and t0 = 08:30 h to tend 08:50 h (c).…”

Identification and classification of host cell proteins during biopharmaceutical process development

“…However, to date, only a few published works have linked upstream and downstream studies together. 16,17,20,21 In a previous paper, 22 we have shown that upstream operating conditions, including culture duration, that are optimized for higher levels of antibody expression, can result in decreased product quality. We have shown the effects of culture duration and upstream harvest time on antibody titer, as well as on product monomer levels and the amount of HCPs present in purified material, which highlighted that culture duration should not be extended purely for the purpose of expressing more antibodies.…”