Most recombinant protein production processes with microbial cell lines like E. coli have traditionally used complex media made up of tryptone and yeast extract to provide the necessary nutrients for cell health and productivity. However, variations in the complex media and potential impurities in different batches can cause serious problems during a bioprocess. For example, amino acid starvation can cause metabolic stress and transcriptional reprogramming to E. coli, leading to a downregulation of genes associated with protein synthesis and upregulation with genes involved with amino acid biosynthesis and cell survival. A recent solution to combat these problems is to use chemically-defined (also known as CD or synthetic) media, which can be optimized and supplemented to prevent nutrient depletion. As such, it is very important to the production of heterologous proteins and therapeutic monoclonal antibodies that we better understand amino acid feed supplementation during a process.

A team from the Indian Institute of Technology has conducted a study using spent media analysis, transcriptomics, and proteomics to optimize the amino acid supplementation strategy. The researchers used an E. coli BL21 strain to express a 13.57 kDa protein multimer inclusion body. For the experiments, cells were grown in 250 mL shake flasks and 1.3 L bioreactors with samples taken hourly to measure cell density, glucose (supernatant run by ion exclusion chromatography), and amino acids (derivatized supernatant run on a UPLC). Harvested samples were also run by HPLC for titer determination, gene expression (RNA-seq), and proteomic analysis (LC-ESI-TOFMS).

The initial shake flask studies revealed a significant difference in cell density between non-supplemented and amino acid-supplemented cultures of CD media. In the supplemented cultures, the cell densities were 50-100% higher, depending on the amino acids’ supplemented time point and concentration. To correspond with the higher cell densities, glucose was consumed faster in all the amino acid-supplemented cultures compared to the non-supplemented cultures. Accordingly, the presence of amino acids led to a faster synthesis of cellular components and higher glucose consumption.

Additionally, protein yield was more than 8-times higher in cultures with at least 5 mM of all 20 amino acids compared with the non-supplemented cultures. Spent media analysis of the amino acid consumption revealed that the first three amino acids to be consumed completely were serine, aspartic acid, and glutamic acid. In fact, these three amino acids were all depleted before protein induction with specific consumption rates of more than 0.9 mmol·gDCW-1·hr-1.

The consumption rates were used to design amino acid supplements for both growth and production phase needs. In the growth phase, serine, aspartic acid, glutamic acid, threonine, and proline were used. In the protein production phase, cysteine, methionine, leucine, and alanine were added. In a series of shake flask experiments, a supplementation protocol including 5 mM of the growth phase blend added at inoculation followed by 2.5 mM of both the growth phase blend and the protein production blend resulted in the highest protein yield. When the same media supplementation strategy was applied to the bioreactor, the supplemented cultures produced 40% more total protein compared to the control. Also, they had a 34% higher product yield (i.e., the protein produced per gram dry cell weight) compared to the control. Additionally, the transcriptomic and proteomic analysis showed the upregulation of amino acid biosynthesis-associated genes in the control cultures and downregulation of the supplemented cultures’ metabolic stress response.

With growing attention toward accelerating therapeutic discovery and production, microbial platforms will continue to be used because they are easier to work with and engineer than mammalian cell lines. The study described here adds to the growing body of research that shows the importance of supplementing synthetic media with amino acids during recombinant protein production. 908 Devices’ newest product, the Rebel, has simplified the ability to monitor amino acids at each phase. One day, hopefully soon, process development labs will no longer need to rely on time-consuming and expensive core labs in the production of recombinant proteins.

By, Glenn A. Harris