Long Peptide or Protein?
As the length of therapeutic peptides increases, so too do the synthetic challenges, as described by Hazel Moncrieff, Ph.D., senior group leader, GMP peptides, Almac Sciences. Errors in amino acid addition to a growing peptide strand are compounded as the peptide lengthens and can greatly compromise the quality of the final product.
“The coupling efficiency at each synthesis step must be extremely high,” resulting from a robust and reproducible synthetic chemistry process, said Dr. Moncrieff. She described Almac’s expertise in producing long peptides and the company’s ability to synthesize peptides greater than 100 amino acids in length routinely. She also reported the successful production of peptides as large as 200 amino acids using solid-phase synthesis.
A key factor in producing long peptides is resin selection to optimize solubility of the forming peptide and minimize aggregation. Another important factor is post-synthesis product characterization and the need for high resolving analytical methods such as UPLC and LC/MS to differentiate full-length peptides from impurities, which are mainly shortened peptides that lack the full complement of amino acids, and which typically elute together with the full-length molecule on separation.
Almac is pursuing in-house research to develop purification technology that Dr. Moncrieff believes will enable production of even longer peptides using solid-phase synthesis. The company is currently testing these new LC-based techniques in small-scale development work, and Dr. Moncrieff reported preliminary findings that demonstrate their ability to increase product yield.
Commenting on industry trends in general, Dr. Moncrieff described an increase in project complexity, with customers requesting the production of both longer peptides and more multicomponent products, in which the API contains a mixture of multiple peptides.
“We are also seeing an increase in the breadth of projects,” she said, referring to growing interest in Almac’s rapidd™ service package that integrates chemical and process development and analytics for API synthesis, drug production, preclinical safety studies, and testing and documentation to support regulatory filings.
The presentation by peptide manufacturer Bachem highlighted the increasing complexity of peptide-based medicines. Lester Mills, chief marketing officer, credited improved analytical techniques amenable to batch processing, mainly UPLC, with providing an enhanced capability to resolve impurities and, thereby, improve process efficiency for the production of longer peptides with more challenging sequences.
“We once thought of the synthesis of proteins as being in the realm of recombinant technology, and peptides in the realm of chemical synthesis,” said Mills. “But these lines are blurring.” With the ability to make peptides of 100 amino acids and longer, “we are getting into the realm of making small proteins” using solid-phase peptide synthesis techniques.
As the frontiers of synthetic chemistry advance, “we can now synthesize peptides we previously could not,” added Mills. As an example, he points to relaxin, a member of the insulin family, which Bachem now offers in its catalog.
“The nature of the sulfur bridges in relaxin make it very challenging to synthesize,” Dr. Mills explained. Other advances that are creating new opportunities for peptide therapeutics include novel drug delivery strategies and the development of therapeutic peptide conjugates. Bachem is seeing an increasing number of projects involving conjugates that combine peptides with small molecules, cytotoxic compounds, or oligonucleotides, for example, reported Dr. Mills.
In June, SpheriTech introduced SpheriTide™, a polymer support for solid-phase peptide synthesis. These polymeric microspheres are composed of peptides, specifically ε-lysine residues cross-linked with multi-functional carboxylic acids.
The first version of SpheriTide offered commercially uses sebacic acid as the cross-linking agent, which forms what the company describes as a “quasi-homogeneous peptide gel network with excellent salvation characteristics, akin to those of the growing peptide chains.”
In the SpheriTide polymer, amide bonds form between the α carboxyl and ε amino groups of lysine, whereas typical peptide bonds form between the α carboxyl and α amino groups of two linked amino acids.
The relatively low cost of poly-ε-lysine, an FDA-approved food ingredient that is commercially available at high purity in bulk scale—produced by bacterial fermentation—gives SpheriTide “the potential, with economies of scale, to be cheaper than polystyrene,” said Don Wellings, Ph.D., founder and CEO of SpheriTech.
He described the ability of the resin’s microporous polymers to swell and solvate and to provide many of the favorable qualities of traditional polystyrene-based support materials. Compared to some new, more hydrophilic support materials, such as polyethylene glycol-based resins, SpheriTide microspheres are not sticky and do not adhere to reactor walls. The cross-linked poly-ε-lysine is biodegradable, with complete enzymatic degradation yielding the polymer’s amino acid components.
The new solid support has resulted in improved product quality and quantity compared to traditional methods for all test peptides synthesized to date by SpheriTech, Dr. Wellings reported.