Real‐time quantification and supplementation of bioreactor amino acids to prolong culture time and maintain antibody product quality

Abstract

Real‐time monitoring of cell cultures in bioreactors can enable expedited responses necessary to correct potential batch failure perturbations which may normally go undiscovered until the completion of the batch and result in failure. Currently, analytical technologies are dedicated to real‐time monitoring of bioreactor parameters such as pH, dissolved oxygen, and temperature, nutrients such as glucose and glutamine, or metabolites such as lactate. Despite the importance of amino acids as the building blocks of therapeutic protein products, other than glutamine their concentrations are not commonly measured. Here, we present a study into amino acid monitoring, supplementation strategies, and how these techniques may impact the cell growth profiles and product quality. We used preliminary bioreactor runs to establish baselines by determining initial amino acid consumption patterns, the results of which were used to select a pool of amino acids which gets depleted in the bioreactor. These amino acids were combined into blends which were supplemented into bioreactors during a subsequent run, the concentrations of which were monitored using a mass spectrometry based at‐line method we developed to quickly assess amino acid concentrations from crude bioreactor media. We found that these blends could prolong culture life, reversing a viable cell density decrease that was leading to batch death. Additionally, we assessed how these strategies might impact protein product quality, such as the glycan profile. The amino acid consumption data were aligned with the final glycan profiles in principal component analysis to identify which amino acids are most closely associated with glycan outcomes.

Productivity improvement and charge variant modulation for intensified cell culture processes by adding a carboxypeptidase B (CpB) treatment step

“…Extending the duration of the cell culture production phase by cell line engineering, medium development and bioreactor optimization are common strategies to improve titer of fed‐batch processes (Druz et al, 2013; Fan et al, 2015; Powers et al, 2019; Wurm, 2004). Regardless of cell culture production duration, each production batch requires similar cell growth time to peak VCD, similar turnaround time between batches, and similar costs for the batch setup including seed expansion, medium preparation, and bioreactor operation (Chen et al, 2018; J. Xu, Xu, et al, 2020).…”

Spectroscopy integration to miniature bioreactors and large scale production bioreactors–Increasing current capabilities and model transfer

“…Furthermore, there has been a huge drive in the pharmaceutical industry to adopt process analytical technologies (PAT) as a result of the FDA PAT Initiative 8 to enhance monitoring and control of processes. PAT, such as Raman [9][10][11] and NIR, 12 are being implemented for on-line monitoring in large scale vessels as they offer a non-destructive approach to measuring multiple analytes simultaneously. However, their use at small scale is limited due to the headspace requirements and capital costs associated with implementing for a large number of vessels.…”

Process analytical technology for on‐line monitoring of quality attributes during single‐use ultrafiltration/diafiltration

“…The three SEC‐based methods were developed as a PAT tool kit, each designed to be fit‐for‐purpose and relatively simple, which aligns with the need for robustness in PAT, and to maximize the value of the data generated. Each method was optimized to obtain mAb purity and excipient concentrations over the wide range of protein concentrations and buffer matrix conditions that a UF/DF process covers (Baek et al, 2019, 2019; Le et al, 2014; Powers et al, 2019). These PAT methods can accurately quantitate mAb purity and the concentration of the excipients, histidine and arginine without the need of chemical tag.…”