Identification of Autocrine Growth Factors Secreted by CHO Cells for Applications in Single-Cell Cloning Media

Abstract

Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium.

A modular and adaptive mass spectrometry-based platform for support of bioprocess development toward optimal host cell protein clearance

“…This comprehensive universal CHO (or any other host organism) library can also be shared across academic and industry users to facilitate HCP identifications. 28,29 Using the DIA method, scientists can not only quantify residual HCPs in a reliable manner (Fig. 6, Table 1), but also gain process knowledge and generate a historical databank that help understand the efficiency of different chromatography steps for successful clearance of specific HCPs.…”

Exploring Packaged Microvesicle Proteome Composition of Chinese Hamster Ovary Secretome

“…A few efforts have been made to reveal the proteome of CHO spent-media [4,6,7]; however they have never been focused to identify and differentiate packed vs. non-packed proteins though their purpose of origin and mechanism of action towards impacting cell growth and recombinant protein production could be immensely distinct. Besides, differentiation between proteins secreted from healthy cells vs. leaked from damaged or dead cells from packed in microvesicles is also extremely important in designing downstream processing strategies and ultimately to better understand secretome.…”

“…For example, ~88% of the identified proteins in suspension culture in the spent-media of CHO cells were categorized as intracellular and non-secretory by Lim et al and ~78% by Valente et al, although the viability of culture under investigation was above 95% [4,24]. This is mainly because majority of the studies designed to identify secreted proteins in media have been able to remove only large sized microvesicles (centrifuge sample ≤10000 rpm or filter with 0.45 µm filter) leaving the high proportion of small microvesicles (~200 nm), including exosomes, in the culture media.…”

“…For example, a number of growth-regulating factors (such as Fibroblast Growth Factor (FGF), Hepatocyte Growth Factor (HGF), Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor C (VEGF-C) and transferrin) have been identified in spent-culture media of CHO cells, whose supplementation in the culture media enhances the cell growth in culture (~48%) even at low cell density and improves the performance of production culture [4]. On the other hand, a number of proteolytic enzymes (matrix metallopeptidase 3 (MMP3), MMP10, MMP12 and cathepsin-B) have been identified in culture media which may pose risk for proteolytic-degradation of the product leading to low yield from production batches [5,6].…”

“…Majorly there are three sources for these type of peptides/proteins in the spentmedia, a) proteins/peptides directly secreted by the healthy cells into the culture media, b) proteins/peptides packed into microvesicles (like exosomes (30-100 nm), microvesicles (100-1000 nm)) for transducing signal to other cells in the culture and/or c) proteins/peptides leaked into the culture media from damaged/dead cells. To date, a number of studies have been performed to identify spent-media proteome [4,6,7], however they were never designed to identify and differentiate proteins directly secreted/leaked from the cells from packed in microvesicles. Microvesicles have already been shown to increase or reduce cell proliferation and hence have capabilities to regulate bioprocess-related cellular phenotypes (like cell growth, yield and product stabilization) in production culture [8][9][10].…”