Speciality Chemicals Magazine SEP / OCT 2024 | Page 28

metabolites and truncated forms of the recombinant target . 5 - 7

Endotoxins also pose a significant challenge when producing recombinant biologics using E . coli , because they can be difficult to remove during post-processing without adversely affecting target molecule recovery . Moreover , this contamination can render the end product unusable for its intended application . Effectively removing host impurities from the target protein is therefore an important step in biomanufacturing .

P . pastoris is able to secrete recombinant proteins into the extracellular environment while secreting low amounts of native proteins , and it does not produce endotoxins . 8 This greatly simplifies downstream processing ( DSP ) and makes it a promising host for biopharmaceutical production .

Ingenza ’ s inGenius * CMC platform offers scalable systems for DSP , such as protein purification methods with low cost of goods , and analytical support for characterising drug substances and impurities . In addition , Ingenza is exploiting P . pastoris ’ lack of endotoxin secretion to explore its potential for expressing a panel of antigens that could be used to develop novel vaccines against sarbecovirus , aiding pandemic preparedness .

Overcoming challenges in manufacturing

Achieving high recombinant protein levels in a bioprocess starts with the construction and isolation of a manufacturing strain . On the other hand , overexpression of a secreted protein can cause a bottleneck in the secretory pathway , resulting in overall lower protein yields . Ingenza owns a collection of genetic elements and signal peptide sequences that overcome these challenges by enabling strain optimisation for the efficient expression and secretion of a wide range of recombinant proteins . P . pastoris can grow on different carbon sources and has the ability to operate in both methanol-induced and methanol-free bioprocesses . Ingenza has capitalised on this by developing superior bioprocesses divided into two phases , reducing competition for carbon between growth and protein production to maximise yields .

One carbon source , such as glucose or glycerol , is initially used to reach very high cell densities without recombinant protein production . This is then switched to a methanol feeding phase , where a very low concentration of methanol is used to trigger methanol-inducible alcohol oxidase I-driven recombinant expression of a target protein .

Ingenza has also developed methanol-free bioprocesses , relying on constitutive or de-repressed promoters , overcoming operational drawbacks associated with the use of methanol , such as high oxygen requirements and heat production , as well as increased costs .

An ideal host

Antimicrobial peptides are naturally occurring molecules with intrinsic antimicrobial properties against a wide range of microorganisms , including bacteria , fungi , viruses and even some parasites . This innate toxicity and natural susceptibility to proteolytic degradation make the recombinant expression of antimicrobial peptides difficult , often resulting in low yields . 9

Ingenza was able to identify P . pastoris as the ideal recombinant host for expression of a novel antimicrobial peptide by testing a panel of inGenius CMC suite microbial host organisms . A manufacturing P . pastoris clone was isolated using Ingenza ’ s visABLE * high-throughput screening platform , enabling the production of grams of the antimicrobial peptide for subsequent toxicology studies .

Conclusion

Selecting an appropriate host organism is possibly the most crucial aspect in the development of a successful biomanufacturing process . P . pastoris is increasingly becoming a popular choice for the recombinant expression of a wide range of proteins due to its versatile genetic toolkit and ability to efficiently produce high-value proteins .

Ingenza has developed optimised P . pastoris strains and bioprocesses for recombinant protein production by coupling automation with strain construction , HTP screening and upstream and downstream processing capabilities . This allows tailored P . pastoris platforms and bioprocesses to be engineered for a wide range of applications spanning healthcare and sustainability . ● * inGenius and visABLE are registered trademarks of Ingenza

28 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981