Using Modified Amino Acids to Simplify Fed-Batch Bioprocessing

With the use of modified amino acids, MilliporeSigma, a Merck KGaA company, has developed a means to simplify the fed-batch process in biomanufacturing. Merck won the 2018 CPhI Pharma Award for Excellence in Pharma: Bioprocessing and Manufacturing for its simplification of the fed-batch process. Pharmaceutical Technology spoke with Andrew Bulpin, head of Process Solutions at MilliporeSigma about their method.

PharmTech: Can you give an overview of your technique/method for simplifying fed batch processes using modified amino acids?

Bulpin: L-tyrosine is a key amino acid for both cellular metabolism and protein synthesis. Its depletion in fed-batch processes has been correlated with a drop in specific productivity and with protein sequence variants. This critical amino acid presents an extremely low solubility, especially at neutral pH.

L-cysteine is a sulfur-containing amino acid which is oxidized to the dimer l-cystine in the presence of air, oxygen or metal containing catalysts such as copper. Whereas L-cysteine is freely soluble, l-cystine has a reduced solubility in water and often precipitates in neutral pH feeds.

To overcome these limitations, common fed-batch processes use highly concentrated alkaline feeds, which create the need for complex control strategies to minimize pH spikes during feed additions. In order to remove this alkaline feed, tyrosine and cysteine were chemically modified to phospho-L-tyrosine disodium salt and S-sulfo-cysteine sodium salt, respectively. The combination of both chemicals allows the integration of both amino acid sources in highly concentrated, neutral pH feeds and thus provide a unique way to simplify fed-batch processes by enabling the development of stable and neutral pH main feeds.

PharmTech: How have you modified the amino acids and how do they function in the fed batch process?

Bulpin: We primarily added reactive functional groups to tyrosine and cysteine respectively to improve solubility (tyrosine) and stability against oxidation (cysteine) at neutral pH and room temperature.

PharmTech: What are the key benefits to simplifying fed-batch processing?

Bulpin: The key benefit to simplifying fed-batch processing is the possibility to include cysteine and tyrosine in highly concentrated neutral pH main feeds. Eliminating the need for a separate alkaline feed for cysteine and tyrosine reduces risk for contamination and eliminates temperature and pH ‘shock’ to the producing cells during the addition of the feeds.

PharmTech: Would using these modified amino acids require modification of quality analysis techniques/methods/sensors?

Bulpin: No, the modified amino acids and the resultant drug product (monoclonal antibodies [mAb]) are able to be tested using standard, current methods. No difference was found between simplified and standard fed-batch processes, indicating that the use of tyrosine derivatives in feeds did not induce any detectable modification on the mAb. We have demonstrated that modified amino acids can be used successfully in highly concentrated feeds to improve the next generation of fed-batch processes.