Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling

Abstract

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data is used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10 L bioreactor model of Lonza's large-scale (up to 20,000 L) fed-batch cell culture processes.Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10 L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10 L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines' performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements.Keywords: Cell line development, Chinese hamster ovary cells, whole cell MALDI-ToF mass spectrometry, PLS-DA modelling, cell line prediction 3 IntroductionThe majority of recombinant protein biopharmaceuticals are produced from cultured mammalian cells (Walsh, 2010), with the most commonly used industrial mammalian cell host being the Chinese hamster ovary (CHO) cell (Kim et al., 2012). Despite the development of high throughput methods that allow the screening of many recombinant cell lines to isolate those with desirable phenotypes (e.g. high growth and productivity), the ability of such methods to select or predict the performance of a given cell line at manufacturing scale remains limited with the best cell lines at a manufacturing scale often distributed across the phenotypic performance of cell lines at smaller-scale (Porter et al., 2010a;Porter et al., 2010b). As a result, early use of a simple productivity based approach does not necessarily allow the identification of high producers in the population of cell lines, and some potentially high producers are discarded early in the process (Porter et al., 2010b). In order to address this issue, a number of proteomic, transcriptomic and metabolic based studies have now been undertaken and the subsequent data used to develop models to predict the phenotype of a given cell line (e.g. Clarke et al., 2011;Clarke...

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