Using biological expression systems to manufacture therapeutic peptides

A proprietary biological expression system to manufacture therapeutic peptides is one of the latest CSIR inventions in life sciences. These peptides are generally manufactured by pharmaceutical companies through chemical synthesis and this route can be complex, costly and inefficient. Globally, the use of biological expression systems is finding favour as a low-cost, environmentally friendly alternative to traditional chemical processes. The HaloBiologixTM technology is being further developed at the CSIR and incubated as a potential new start-up venture.

HaloBiologixTM is the brand name given to a grouping of technologies used for the production of pharmaceutically
relevant peptides and proteins.  The name was derived from the development of a proprietary expression host,
Bacillus halodurans Alk36, as a potential expression system for the production of therapeutic peptides.  The proprietary Bacillus halodurans Alk36 strain of bacteria was found to produce flagellin protein at high levels.

The flagellin protein is the main building block of flagella, a surface-exposed appendage, used for motility. The ability to produce large numbers of flagella (and flagellin) was utilised to develop a surface display system. Surface display technologies are used in applications such as biosensors,  vaccines, nanotechnology, biosorbants, and whole cell
biocatalysis. Furthermore, through genetic inactivation of genes on the chromosome involved in the production of flagella, the flagellin secretion pathway, also known as the type III secretion system, was genetically engineered to
successfully secrete recombinant therapeutic peptides into the supernatant.

Dr Michael Crampton, a senior researcher at the CSIR says: “Currently, therapeutic peptides are produced using chemical
synthesis procedures. This process is expensive and results in the production of large quantities of toxic waste. The number of steps required for the synthesis of larger peptides (>35 amino acids) is also excessive and adds to the high cost of production. Through the use of the CSIR-patented expression technologies, we hope to produce such peptides at a much lower cost. This will result in drugs being made available to lower income patients, especially in Africa.

“South Africa’s entire active pharmaceutical ingredient (API) requirements for the antiretroviral treatment programmes
are imported, mainly from India and China, but also from other countries. We cannot continue to rely on other countries to supply products for which we require a high level of quality and security of supply for the antiretroviral treatment programme. Secondly, the pharmaceutical sector has for good reason been identified as one of four lead sectors which could be used to diversify South Africa’s industry (away from its predominantly resource-based nature) and to address a worsening trade balance problem (present trade imbalance for the pharmaceutical sector alone is R11.8 billion),” says Crampton.

In further genetic modifications of the Bacillus strain involving the inactivation of five protease genes, protein secretion was enhanced, giving rise to peptide concentrations in the range of mg/L as opposed to the previous μg/L. Actual concentrations vary depending on the nature of the peptide. The peptides are secreted into the medium as protein fusions with built-in cleavage sites and affinity tags, thereby facilitating purification.  The cleavage sites are amino acid
sequences which are recognised by specific proteases and allow for the separation of the carrier protein from the therapeutic peptide of interest. The affinity tag is also a specific amino acid sequence, six histidine amino acids, and facilitates the binding of the produced protein to a metal based affinity resin for purification (IMAC, immobilised metal
ion affinity chromatography).  Peptides of up to 80 amino acids and small proteins have been secreted in this way without the need for expensive inducer chemicals.

In 2009, the Cape Biotech Trust (now Technology Innovation Agency) invested in the development of the expression
system. The CSIR agreed to incubate the HaloBiologixTM technology with a view to spinning it out as a new start-up venture to commercialise the biological route to recombinant peptide production.  In doing this, the CSIR team is
collaborating with the chemical engineering department of the University of Cape Town and the Technical University of Berlin.

“Recently, government has called for the rapid implementation of sectorial strategies and for leveraging of the state’s
pharmaceutical procurement programme in order to stimulate the local production of APIs. Finally, new ARV drugs, such as Enfuvirtide, that are highly effective, are also very costly and as such will forever be out of reach of African patients unless alternative, cost-effective manufacturing methods are found that will lower the cost of production,” adds
Crampton.

Evaluating the proprietary HaloBiologixTM technology has resulted in the successful expression of antigenic peptides
(useful for vaccine development), anti-microbial peptides (useful as a topical antibiotic) and antiviral pre-curser peptides (useful in antiretroviral therapy). Many of these peptides need posttranslational modifications such as amidation and/or acetylation to improve efficiency. Chemical and biological approaches to address this are being evaluated and developed.

In addition to the biological production of peptides, a number of alternative applications for the technology are being
explored. These include vaccine development for both veterinary and human applications; surface display of peptides for applications in metal binding; biocatalysis, biosensors and synthetic biology applications in artificial scaffolding
development and nanotechnology.

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