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Feature Articles : Jul 1, 2012 (Vol. 32, No. 13)

Reducing Peptide Synthesis Costs

  • John Russell

Peptides have long floated in a kind of no-man’s land between small molecules and proteins. Their high specificity and low tox profiles are attractive, but high production costs along with delivery and bioavailability issues have tempered drug industry enthusiasm.

Recently, the broad rush toward biologics has rekindled biopharma’s interest in peptides and prompted peptide suppliers to seek new ways to trim production costs and tackle longer, more complex peptides.

While Fmoc chemistry remains the backbone for most peptide synthesis, a diverse array of process optimizations and advancing instruments are helping peptide suppliers wring out production costs, improve purification, and reduce solvent waste. Bachem, for example, reported that switching from HPLC to Ultra HPLC reduced the time required for one process from 50 minutes to eight minutes and improved results. Recombinant processes are making similar strides.

Accurately ascertaining the peptide market size is difficult; most estimates converge around $800–$900 million for the total peptide API market, with roughly half of that available to noncaptive suppliers.

“Perhaps 20 percent of all products approved this year will be biologics, including six or more peptide products,” said Rodney Lax, Ph.D., senior director of business development, The PolyPeptide Group. “There is enormous interest in peptides.”

Peptides International (PI) recently entered a partnership with API supplier Peptisyntha (member of the Solvay Group) for the production of research-grade peptide APIs. “It’s a nice strategic partnership and shows the confidence they have in our research capabilities,” said Mike Pennington, Ph.D., president and COO of Peptides International.

The company emphasizes speed and quality and uses a variety of robotic systems to speed throughput, according to Dr. Pennington. “Most of these are parallel peptide synthesizers that make 50 micromoles to about 2 millimoles using, for example, 12- and 6-channel synthesizers,” he said.

Demand from biopharma is growing, he added. “There’s a tremendous thrust for new peptide research, especially with regard to oncology, because these cell-penetrating peptide (CPPs) motifs turned out to be incredibly useful tools for delivering drugs to the inside of cells.”

He cited antineoplastic drugs such as doxcorubicin, cisplatin, and chlorambucil, and their ability to be selectively delivered to tissues that have cancerous or neoplastic growth via these novel CPPs. “I definitely believe the API peptide manufacturing sector will have substantial growth. You can see the number of projects that are in Phase II and III clinical development.”

Nevertheless downward price pressure characterizes the research peptide market, partly because it is less subject to regulatory control and partly because of global suppliers in markets with low labor costs. Dr. Pennington suggests a buyer-beware policy on quality.

Hybrid Approaches Proliferate

One continuing trend is blurring of the line between solid-phase peptide synthesis (SPPS) and liquid- (or solution-) phase peptide synthesis (LPPS) as companies seek to combine the benefits of both approaches, noted Barry O’Connor, Ph.D., research scientist, Sekisui Medical, who presented the company’s Molecular Hiving (MH) technology at the recent “TIDES” conference.

Traditionally LPPS offers better economies of scale for large quantities, while SPPS is faster and well suited for synthesizing longer peptides. Sekisui’s MH technology is hydrophobic tag-assisted LPPS that integrates the key advantages of SPPS for practical and highly efficient preparation of peptides. “It is applicable for the development of peptides ranging from short to long and can be easily transferred from small (g) to large scale (kg),” said Dr. O’Connor.

In brief, peptides attached to the hydrophobic tags form concentrated reaction fields as reverse-micelles. After each reaction cycle, adding more solvent causes the growing peptide chains in the micelles to precipitate, simplifying removal before proceeding to deprotection and the next cycle.

“An important advantage,” Dr. O’Connor explained, “is that it allows us to carry out efficient and cost-effective production of short and long peptides by using an inexpensive achiral hydrophobic tag in place of an expensive solid support. Once the tag is in place, simple peptide coupling/deprotection chemistry is utilized to deliver the desired peptides in excellent yield and high purity.”

To demonstrate MH’s strength, Sekisui synthesized the direct thrombin inhibitor 20 mer peptide, bivalirudin, using both MH technology and SPPS for comparison. MH, after 42 steps, resulted in a total yield of 71% with crude purity of 83%, significantly superior to SPPS, which delivered a purity of only 60%. Moreover, MH’s costs were substantially less than those for SPPS, noted Dr. O’Connor.

Another company attacking production costs and purity by blending SPPS and LPPS concepts is Ajinomoto. Daisuke Takahashi, chief, Research Institute for Bioscience Products and Fine Chemicals at Ajinomoto, described the company’s new approach.

Ajinomoto’s AjiPhase process “overcomes the disadvantages of traditional LPPS yet retains the benefits of lower cost, higher quality, and easier scale-up compared to SPPS,” said Takahashi. “We have applied this technology to the synthesis of a variety of peptides including larger than 40 mers, cyclized, and conjugated peptides in high volumes. The significant reductions in production costs and purification load compared to SPPS have been demonstrated.”

AjiPhase’s liquid-phase advancements—including patented anchors, unique and efficient deprotection, and scavenger agents—eliminate the isolation, crystallization, and purification steps between couplings, according to Takahashi. The higher purity of crude peptide product created by AjiPhase lessens the purification load and helps reduce the development time normally associated with LPPS, while generating a greater yield of higher-purity peptides, he said.

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.

IR Heating

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.