Tuning monoclonal antibody galactosylation using Raman spectroscopy‐controlled lactic acid feeding

Abstract

A key aspect of large-scale production of biotherapeutics is a well-designed and consistently-executed upstream cell culture process. Process analytical technology tools provide enhanced monitoring and control capabilities to support consistent process execution, and also have potential to aid in maintenance of product quality at desired levels. One such tool, Raman spectroscopy, has matured as a useful technique to achieve real-time monitoring and control of key cell culture process attributes. We developed a Raman spectroscopy-based nutrient control strategy to enable dual control of lactate and glucose levels for a fed-batch CHO cell culture process for monoclonal antibody (mAb) production. To achieve this, partial least squares-based chemometric models for real-time prediction of glucose and lactate concentrations were developed and deployed in feedback control loops. In particular, feeding of lactic acid post-metabolic shift was investigated based on previous work that has shown the impact of lactate levels on ammonium as well as mAb product quality. Three feeding strategies were assessed for impact on cell metabolism, productivity, and product quality: bolus-fed glucose, glucose control at 4 g/L, or simultaneous glucose control at 4 g/L and lactate control at 2 g/L. The third feeding strategy resulted in a significant reduction in ammonium levels (68%) while increasing mAb galactosylation levels by approximately 50%. This work demonstrated that when deployed in a cell culture process, Raman spectroscopy is an effective technique for simultaneous control of multiple nutrient feeds, and that lactic acid feeding can have a positive impact on both cell metabolism and mAb product quality.

Exometabolome profiling reveals activation of the carnitine buffering pathway in fed‐batch cultures ofCHOcells co‐fed with glucose and lactic acid

“…This study compared the metabolic profiles of two experimental treatments of mAb‐expressing GS‐knockout CHO‐K1 cells grown in bioreactors: cells grown with a controlled lactic acid feed designed to maintain lactate at 2 g/L (Lactic acid feed), and cells grown without a lactic acid feed (Control). Feeds were controlled using a feedback control loop informed by Raman spectroscopy (for more detail on this work, please refer to Eyster et al 11 ). For both treatments, glucose was maintained at 4 g/L using a controlled glucose feed.…”

“…Details of the model cell line and bioreactor operation used in this study can be found in Eyster et al 11 Briefly, a glutamine synthetase (GS)‐knockout CHO‐K1 cell line was thawed into a proprietary, chemically‐defined seed medium and passaged in shake flasks. At the N‐1 stage, three 3 L Applikon bioreactors with a custom‐built control system were inoculated to a target viable cell density of 0.4 × 10 5 cells/mL at a working volume of 2 L. After 4 days of culture, cells from the two reactors with the highest viable cell count (VCC) were pooled to inoculate eight 3 L production bioreactors to an initial working volume of 1.9 L and target viable cell density of 1.0 × 10 5 cells/mL (one reactor subsequently failed due to a faulty DO probe).…”

“…Samples were manually collected daily for measurement of cell concentration and viability (Vi‐Cell XR, Beckman Coulter, Pasadena CA), pH/DO/dCO 2 (RapidLab 248 Blood Gas Analyzer, Siemens, Germany), and osmolality (A 2 O, Advanced Instruments, MA). Samples were typically taken before feeding as described in Eyster et al 11 One set of daily filtered supernatants were used for titer and metabolite analysis (Cedex BioHT, Roche, Switzerland; YSI 2900, YSI, OH) (measurements derived from these daily offline samples were subsequently used in Eyster et al 11 ), and another smaller set was designated for exometabolomics analysis and sent to Cellzome GmbH, GlaxoSmithKline R&D, Meyerhofstrasse 1, 69,117 Heidelberg, Germany. The samples for exometabolomics analysis were taken on days 0, 5, 7, 10, 13, 15, and 17 and were prepared by first a 3 min centrifugation at ×13,300 g and 4°C, and a second centrifugation of 0.7 mL aliquots of supernatant using 0.2 micron Spin‐X tubes at ×13,300 g and 4°C.…”

“…Details of the use of Raman for dual nutrient control and the necessary chemometric model development may be found in Eyster et al 11 Briefly, two multichannel RamanRxn2 analyzers (Kaiser Optical Systems, Ann Arbor, MI) with a 785 nm laser and bIO‐Optic immersible optics were used to monitor glucose and lactic acid, with each Raman unit capable of monitoring up to 4 reactors at a time. Based on the number of spectra acquisitions and acquisition time (along with the limitation imposed by the instrument requiring sequential acquisition for each probe), a glucose/lactate prediction was generated from a SIMCA‐Q chemometric model once per hour for each reactor.…”

“…Alternatively, the pH can be lowered to reduce lactic acid production 10 . Recently, we reported using a lactic acid feed as a process lever for reducing ammonium accumulation and increasing mAb galactosylation 11 …”