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.