Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Abstract

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples. 10,11 , and the presence of various glycan species has been linked to bioprocess parameters such as feed strategy, pH, and temperature 12. The effect of the varying media conditions on product quality, indicated by the percentage of the resulting IgG in monomeric form, is also evaluated with Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS) 13,14,15. The charge variant profile represents a number of modifications 16 that

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

“…For amino acid analysis by LC–MS, crude bioreactor media was centrifuged and passed through a 0.22 μm filter. A perchloric acid cleanup was used to remove protein and particulate matter, which involved mixing filtered bioreactor media with 0.4 N HClO 4 at a 1:1 ratio and centrifuging at 1,962 g for 5 min at RT . The clarified media was collected to be analyzed by LC–MS.…”

“…Media samples were run in triplicate, with error bars indicating SD s. Injection order was randomized to eliminate order bias. Additional information on this method can be found in past work …”

“…Samples were analyzed in triplicate at 2.5 μg (2.5 μl at 1 mg/ml) and denatured at 70°C for 10 min with 7.7 mM Iodoacetamide (ThermoFisher Scientific Cat# 90034), nonreducing conditions, or with 30.7 mM DTT (Sigma‐Aldrich Cat# 10197777001), reducing conditions. Relative peak areas were determined using the GXII reviewer software …”

“…The GlycoWorks method used to label and quantitate glycans isolated from the harvested antibodies was described previously. 20 The glycan profiles were only evaluated for the final harvest product for each bioreactor that was run. The data for the principal component analysis was analyzed using Unscrambler X 10.4 (Camo, Oslo, Norway).…”

Multivariate data analysis of growth medium trends affecting antibody glycosylation

“…29 After purification, a Thermo Scientific NanoDrop One microvolume UV-Vis spectrophotometer was used to determine the sample concentration while the protein extinction coefficient of 13.7 at 280 nm for a 1% 29 After purification, a Thermo Scientific NanoDrop One microvolume UV-Vis spectrophotometer was used to determine the sample concentration while the protein extinction coefficient of 13.7 at 280 nm for a 1%…”