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.

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 are 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 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L 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.

An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer.

In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG(4) monoclonal antibody production by GS-CHO cells throughout a state-of-the-art industrial fed-batch culture process. Based on the calculation of a production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy-intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of production. Instead, cell-specific Mab production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole-process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase production of a recombinant protein by a mammalian host cell--where control of productivity is inherently protein product and cell line specific.