Lower Cost Approach
Many complicated peptides are most effectively manufactured using recombinant technology, however the cost can be off-putting. Startup AmideBio has introduced a lower cost approach, BioPure, based on technology licensed from the University of Colorado.
“The work was driven by the need for larger quantities and lower cost of amyloid peptides for our studies on Alzheimer disease,” said Michael Stowell, Ph.D., an associate professor at CU Boulder and AmideBio co-founder & CTO. “What we’ve done is leverage technologies from different fields. It’s more of a hybrid process than a strictly synthetic or recombinant process.”
AmideBio has a library of vectors with specific affinity tags and cleavage tags. The target peptide is introduced into a series of those vectors. They are then screened to select a high-expressing vector to produce large amounts of material from which the fusion construct (peptide and tags) is extracted, explained Dr. Stowell.
The key component of the process “is what we call Cap-Clip (capture and clip) technology. The fusion construct contains the AmideBio cleavage sequence and an affinity tag,” said Dr. Stowell. “What’s important is both tags have orthogonal chemistries for the target peptide. That means the target peptide has completely orthogonal affinities for the bead material compared to the tag and the cleavage sequence—this allows us to achieve very high purity.”
Choosing the correct vector is a quick process, said Dr. Stowell, “a matter of a few calculations and five or ten minutes on the computer. Actually constructing the DNA expression vector is the more time-consuming aspect, but we’ve gotten to the point where from a sequence to that initial expression component is less than a week.”
AmideBio’s method reduces production costs and by its nature also reduces the waste stream (solvents) associated with peptide synthesis. The company currently serves the research market for difficult-to-make peptides that require ultra-high purity such as amyloid peptides, which the company supplies at greater than 99% purity, noted Dr. Stowell. The firm also has an ongoing peptide therapeutics development effort that leverages the technology.
Peptide synthesizer and consumables supplier Protein Technologies (PT) introduced new heating capability based on infrared technology (IR). “This is not just a refinement,” said PT CEO Mahendra Menakuru. “It’s never been done before. Most people have a mindset that only microwave produces rapid heat, but we have chosen to take the IR methodology and make it our rapid heat model.”
In some cases, rapid heat is useful in promoting the synthesis reaction, while at other times it can be detrimental. The new PT feature provides another tool for users. “If you do need rapid heat in tandem for two reaction vessels and different set points, that capability is also available,” said Menakuru.
PT, which focuses exclusively on the peptide synthesis market, offers a diverse line of instruments. PT’s advanced fluidics, including a patented valving system (membranes actuated by air), substantially improve instrument reliability, according to Menakuru.
Bioavailability Challenges Persist
The industry is always looking for new technologies to make synthesis and manufacture more economical, said Dr. Lax of The PolyPeptide Group, “but I think there is some reluctance to move away from current technologies. Fmoc chemistry in general works really well. I think most changes in the near future are going to be made through optimizing the chemistry we have.”
Improving delivery and bioavailability remain pressing needs and areas of active investigation. “Everybody is trying to find peptides that are more potent so the dose is lower, or they are trying to extend the biological half-life—mainly by developing long-acting release methods or conjugating peptides to nonpeptidic moieties that give them more extended biological activity,” according to Dr. Lax.
The obvious one is PEGgylation. This technology has been employed with proteins for a very long time. Dr. Lax notes the first PEGylated peptide (peginesatide from Affymax) was approved this year. “I am sure there will be others,” he said.
However, there are issues with PEGylation. One is the accumulation of PEGs in tissues after chronic administration because it is not easily removed from the body. “We’ll see a move to discover alternative large conjugates that are more amenable to biological degradation,” said Dr. Lax.
Detection of impurities is another challenge. “The peptides we’re making typically have a molecular weight in the range of 2,000–4,000 Daltons and PEGs are typically 20–40K Da. Once you attach a peptide to the PEG, any impurities already in the peptide or generated during the conjugation are masked by the broad PEG peak and cannot be detected analytically by HPLC methods.”
Until recently, said Dr. Lax, the approach to manufacturing PEGylated peptides was to prepare the peptide as purely as possible, demonstrate the high purity analytically, and then PEGylate it. “The logic being that the peptide was pure before I attached the PEG and therefore it must be pure after I attached it. I think the regulatory authorities no longer totally buy into that.”
There is currently no “really good technology for detaching the PEG” and Dr. Lax said he believes we may in the future see PEGs that are reversibly attachable to the peptides or that can be detached using some chemical or enzymatic technology. This would allow us to recover the peptide in order to determine its impurity profile.