Improving Protein Production in CHO Cells - Biopharmaceutical protein manufacture can benefit from using chemically defined feeds in CHO cell lines. - BioPharm International

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Improving Protein Production in CHO Cells
Biopharmaceutical protein manufacture can benefit from using chemically defined feeds in CHO cell lines.


BioPharm International Supplements


Abstract

With the drive to remove serum and products of animal origin from cell-culture media during manufacture of protein-based biologics, plant hydrolysates have become popular as feeds in fed-batch cell culture to boost productivity. However, plant hydrolysates often contain undefined levels of nutrients, which can result in lot-to-lot variation in fed-batch performance and inefficiency. These are disadvantages when consistent protein yields are required from recombinant Chinese hamster ovary (CHO) cell lines. To achieve a more controlled process, one approach is to use chemically defined formulations as feeds containing only a carbon source, amino acids, vitamins, and trace elements. As a test of this approach, we compared cell growth and protein production characteristics of CHO cell lines grown in chemically defined media supplemented with either chemically defined nutrient feed supplements or plant hydrolysates alone, or with nutrient feed supplements.


Invitrogen Corporation
Removing serum and products of animal origin from cell-culture media during production of therapeutic proteins from Chinese hamster ovary (CHO) cells offers improved biosafety and can make regulatory approval smoother. However, not having these supplements in the media can limit CHO cell growth and protein production. One method of improving growth is to add plant protein hydrolysates as a feed because they provide a highly pure source of soluble amino acids, peptides, vitamins, and essential elements for cell culture. Supplementing media with plant hydrolysates has been shown to improve protein production from engineered CHO cells.1,2

The drawback of using plant hydrolysates is that they often contain undefined levels of nutrients and can result in lot-to-lot variation in fed-batch performance and purification inefficiency, which are disadvantages when consistent protein yields are required.3 To achieve a more controlled process, it is important to begin with a base medium for batch culture that does not make feeding strategies more complicated. An alternative approach to using nutrient-rich plant hydrolysates is to use a medium that is specifically designed to promote rapid cell growth and high levels of protein expression for CHO cells as a basal media, and then add chemically defined feeds containing only a carbon source, amino acids, vitamins, and trace elements to replenish the nutrients that are being depleted in the media.4 This article describes the experimental performance of chemically defined nutrient feed supplements compared to plant hydrolysates in a chemically defined medium with a recombinant CHO cell line.

Experimental Approach

Shake Flask Studies


Figure 1
In a first shake flask study (Figure 1), recombinant CHO cells engineered to express a proprietary antibody were inoculated at 3 x 105 cells/mL into 30-mL shake flasks containing CD OptiCHO Medium (Invitrogen, Grand Island, NY) as a base. The cells were incubated at 37 C in 8% CO2, and agitated at 125 rpm. To determine the effect that plant hydrolysates or a chemically defined medium had on protein production, on days 3, 6, and 9, a 10% v/v solution of one of the plant hydrolysates derived from pea, wheat, soy, or rice (Kerry Bio-Science, Rochester, MN) was added at a stock concentration of 100 g/L to the cultures or the media were supplemented with a 10% v/v chemically defined feed, CHO CD EfficientFeed A (Invitrogen, Grand Island, NY). For each flask containing a different type of feed, there were three replicates made and samples were withdrawn for high performance liquid chromatography (HPLC) analysis of IgG concentration from each replicate every 24 hours on days 7–14. In those instances, when cell viabilities dropped below 20%, samples were no longer obtained.


Figure 2
In a second shake flask study to determine if protein production could be increased by using a combination of feeds (Figure 2), 3 x 105 cells/mL were inoculated into 30-mL shake flasks containing the same base medium. They were incubated at 37 C in 8% CO2, and agitated at 125 rpm. On days 3, 6, and 9, a 10% v/v addition of the same solutions of pea, wheat, soy, or rice plant hydrolysates plus a 10% v/v solution of chemically defined feed A were added simultaneously, or a 10% v/v solution of chemically defined feed A was added alone to the cultures. For each flask containing a different feed combination, there were three replicates and samples were withdrawn for HPLC analysis of IgG concentration from each replicate every 24 hours on days 7–14.


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