Microarray-Based MALDI-TOF Mass Spectrometry Enables Monitoring of Monoclonal Antibody Production in Batch and Perfusion Cell Cultures

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July 15, 2016

Authors

Robert F. Steinhoff1, Daniel J. Karst2, Fabian Steinebach2, Marie R.G.Kopp2, Gregor W.Schmidt3, Alexander Stettler3, Jasmin Krismer1, Miroslav Soos4, Martin Pabst1, Andreas Hierlemann3, Massimo Morbidelli2,and Renato Zenobi1

Organizations

Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland
Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland
Bio Engineering Laboratory, Department of Biosystems Science and Engineering, ETH Zurich, CH-4058 Basel, Switzerland
Faculty of Chemical Engineering, University of Chemistry and Technology, 16628 Prague, Czech Republic

Abstract

Cell culture process monitoring in monoclonal antibody (mAb) production is essential for efficient process development and process optimization. Currently employed online, at line and offline methods for monitoring productivity as well as process reproducibility have their individual strengths and limitations. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based on a microarray for mass spectrometry (MAMS) technology to rapidly monitor a broad panel of analytes, including metabolites and proteins directly from the unpurified cell supernatant or from host cell culture lysates. The antibody titer is determined from the intact antibody mass spectra signal intensity relative to an internal protein standard spiked into the supernatant. The method allows a semi-quantitative determination of light and heavy chains. Intracellular mass profiles for metabolites and proteins can be used to track cellular growth and cell productivity.

CovalX Technology Used (Click each option to learn more)

Outcomes

A CHO cell line (secreted IgG1 isotype recombinant mAb) that was stored in liquid N2 was thawed and expanded in suspension for one week. During this week, the cell line was diluted every other day with fresh media. In a spin tube, the batch experiment was inoculated at 1 x 106 cells/mL and then cultured in a humid atmosphere (36.5 °C, 5% CO2) in a shaking incubator (320 rpm). The cells were allowed to expand for another week in a seed bioreactor and then inoculated from a working volume of 40 mL to a 1.5 L perfusion bioreactor that used tangential flow filtration (TFF) in order to retain cells. 1 reactor volume/day was used as the harvest rate and 20 x 106 cells/mL was used as the cell density while the operating parameters were set to 36.5 °C, pH 7.1 and 50% dissolved oxygen tension. Cell density, viability, mAb titer, glucose and lactate concentrations were measured daily. After 6 days, the average cell diameter ewas 12 μM. 5 x 106 cells were spun down (1000xg, 1 min, 4 °C) before the supernatant was collected and stored at -80 °C. Using 1000 μL 10 mM NH4OAc (pH 7.2, 4 °C), the cell pellet was washed and then vortexed. 300 μL of MeOH/H2O (50%/50%, v/v) solution that contained 100 μM 13C-ATP and 100 μM D4-Alanine was added to lysed cells before being vortexed for 30 seconds. 400 μL MTBE (100 μM DSPC) was added and the mixture was vortexed for 30 more seconds. To separate the phases, the samples were centrifuged at 16,000xg for 2.5 minutes at 4 °C. Individual phases were each collected in 1.5 mL tubes and stored at -80 °C.

A matrix was prepared by dissolving 9-Aminoacridine (10 mg/mL) in 100% acetone before removing the insoluble particles using centrifugation (14,000xg, 1 min).Using a volume ratio of 1:10, the aqueous extraction phase was mixed with the 9-AA solution. Sinapic acid (15 mg./mL) was dissolved in 50% aqueous acetonitrile that contained 0.1% TFA. This solution was mixed with the extracellular protein fraction in a ratio of 10:1. DHAP was mixed in a 25:25:50 ratio with acetonitrile, ethanol and water containing 0.1% TFA. The DHAP solution was then mixed with the intracellular protein fraction in a ratio of 10:1.

Using a self-aliquoting sample target, 6 μL of each sample mixture was spotted on a target plate and aliquoted using a slider twice. Intracellular protein analysis performed using a mass spectrometer that had been modified with a CovalX high mass detection system.

Extracellular protein content in the bioreactor did not require prior purification, only a homogenous matrix-analyte spot mixture. This was achieved by using a plasma asher to clean a 4-inch silicon wafer in oxygen plasma before being spin coated in polysilazane solution and cured on a hotplate (135 °C, 4 hours). On top of the polysilazane, positive photoresist was spin coated and microstructured using photolithography and a mask aligner with a transparency mask. Using a reactive ion etcher, the polysilazane was removed in exposed areas and the residual positive resist was removed using a toothbrush and an acetone bath. Using a dicing saw, 75 mm x 26 mm microarray chips were cut from the wafer. From the analysis of these microarrays for mass spectrometry (MAMS), a 23 kDa and 47 kDa fragment of the mAb were detected. Thus, the 23 kDa fragment was assigned the light chain and the 47 kDa fragment was assigned the heavy chain. Data from the MALDI analysis allowed for researchers to determine that the average constant mAb concentration in the perfusion reactor was 0.2 g/L (±0.7 g/L) as well as detect mAb aggregates. The use of MALDI mass spectrometry allowed for the monitoring of monoclonal antibody production in analytes while requiring less sample preparation and analysis time than HPLC-UV methods that are typically used. MALDI mass spectrometry is sensitive to a number of metabolites, however it was especially useful in detecting a high number of phosphorylated species that are otherwise difficult to monitor using HPLC-MS.