Microarray-based MALDI-TOF mass spectrometry enables monitoring of monoclonal antibody production in batch and perfusion cell cultures

Highlights

• Microarray based MALDI-MS for metabolite and antibody monitoring shows excellent results.

• Monoclonal antibody is monitored directly from cell supernatant without prior purification.

• The MALDI-MS method is significantly faster and cheaper compared to HPLC–UV approaches.

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.

Introduction

Monoclonal antibody (mAb) producing cell cultures are indispensable in biopharmaceutical production and their systematic assessment is of great interest to academic and industrial research. MAbs have undergone an impressive development from their first description by von Behring and Kitasato in 1890 to their widespread clinical applications [1], [2]. Today, also smaller recombinant antibody fragments such as fab and scFv, or antibody mimetics, such as designed ankyrin repeat proteins (DARPins) are studied for use in diagnostics and therapy [3], [4]. Three of the top ten drugs by worldwide sales are whole mAbs and consequently, their production processes are of great interest [5], [6]. Industrial monoclonal antibody manufacturing includes several manufacturing steps as reviewed by Wurm in 2004 and Thoemmes in 2010 [7], [8]. Due to the required posttranslational modifications antibodies are commonly expressed in mammalian cell cultures. Typical culture modes are small-scale, batch, fed-batch and perfusion reactors [9]. The different process designs lead to overall significant differences in antibody yield, purity and integrity. In this context, cell culture monitoring is a key element.

Besides the classical process parameters, such as cell viability, oxygen levels, and substrate sensing, more analytical techniques are implemented to support process engineering [10]. State-of-the-art analytical methods for mAb process monitoring are based on charge separation by electrophoresis, such as capillary electrophoresis (CE), isoelectric focusing and size separation by electrophoresis as for example sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). Moreover, there are a number of established high performance liquid chromatographic (HPLC) methods available for protein analysis, including ion exchange HPLC, reversed-phase HPLC and size exclusion HPLC with UV or mass spectrometric (MS) detection [11], [12], [13]. Structural and functional analysis of mAbs is performed by HPLC–MS [14]. Dynamic light scattering and analytical ultracentrifugation are mainly used for mAb aggregation analysis [15]. The list of analytical tools for mAb detection is expanded by very rapid spectroscopic methods, such as FT-infrared spectroscopy, circular dichroism, and Raman spectroscopy for probing the antibodies secondary structure. Metabolite monitoring and profiling is mainly performed using mass spectrometry or nuclear magnetic resonance (NMR) [16].

Another very promising approach for cell culture monitoring is based on matrix-assisted laser desorption/ionization (MALDI) [17], [18], [19], [20]. This soft ionization method allows for the detection of small and large biomolecules directly from complex media or extraction solutions mostly singly charged. The ions can be detected with various detector systems, including multi-channel plate (MCP), ion-conversion dynodes, Faraday cups and pixelated time-to-digital converter [21], [22], [23]. Since ion-to-electron conversion is drastically decreasing with decreasing ion momentum, high mass ions are rather detected by ion-conversion dynodes or pixelated detectors [24], [25]. Generally, MALDI offers a high salt tolerance resulting in extremely short and simple sample preparation steps. Combined with its straightforward data analysis MALDI-MS is an ideal tool for process monitoring. The use of a microarray for mass spectrometry (MAMS) in combination with appropriate internal standards allows to reduce MALDI’s poor reproducibility and to offer semi-quantitative MALDI-MS analysis [26]. Here, we describe a bioreactor monitoring method based on MALDI mass spectrometry using a specifically optimized microarray sample target for mAb quantification and metabolite detection. The method is based on an “all-in-one” extraction step using MeOH/water and methyl-tert-butyl ether (MTBE) as extraction solvents, which enable the analysis of intracellular proteins, metabolites and lipids. To demonstrate the performance of MALDI-MS for this application, a batch and a perfusion reactor were monitored over 6 days, respectively. Intracellular metabolite levels were measured in host cell lysates. An intracellular protein profile was recorded for the batch process and the mAb titer was determined from the intact mAb mass signal intensity as obtained directly from the unpurified supernatant. Results were cross-validated with state-of-the-art HPLC-UV methods. MALDI-MS as an at-line process analytical tool (PAT) proved to be a fast and robust technique.