Neoantigen-encoding peptides often consist of amino acid changes, inserts, and/or deletions, which significantly affect peptide hydrophobicity. These hydrophobic peptides have high tendency to aggregate and be insoluble making them difficult to purify and utilize downstream.
GenScript has achieved a >95% successful synthesis and delivery rate for neoantigen peptides by using a combination of proprietary peptide production chemistries/processes and bioinformatics tools.
What Makes Neoantigen Peptides so Difficult to Produce?
|Length||The isoelectric point (pI) of a peptide chain||Neoantigen peptides range from 8 - 50 amino acids. Peptides over 20 AA are difficult to be synthesized|
|Charge||The isoelectric point (pI) of a peptide chain||Many purification steps only work with specific charges, the further from neutral a peptide is, the synthesis will be more difficult|
|Hydrophobicity||Amino acids which naturally repel water due to their charge||The more hydrophobic a peptide is, the more difficult synthesis and purification will be due to its insolubility in water|
|Yield||The amount of one peptide in weight (mg)||It is very difficult to find a provider who can generate a range of yields for difficult peptides|
|Purity||The percentage of an individual peptide in a solution of peptides||The charge and hydrophobicity (solubility & aggregation) make standard purification difficult|
The Intrinsic Characteristics of Neoantigen Peptides Which Make Them Difficult to Synthesize.
Neoantigen peptides are usually expressed on the cell surface, and arise due to single amino acid deletions, insertions, or substitutions. Because of this, they vary in length, charge, and hydrophobicity, leading to difficulty in synthesis and purification.
How Does GenScript Get Around These Difficulties?
GenScript has developed NeoPre™, a predictive algorithm which is able to determine peptide synthesis difficulty based on sequence alone. NeoPre™ analyzes six intrinsic characteristics of each peptide in order to predict synthetic difficulty, including length, yield, charge (pI), aggregation, hydrophobicity, and purity. NeoPre can then recommend the most efficient approach to successfully synthesizing peptides using one of GenScript’s many synthesis platforms.
|27||20-24 mg||>98%||7.4||24.46296||-0.25||Difficult||Automatic synthesis|
|25||4 mg||>98%||10.2||52.2536||0.65||Difficult||Mankind handling with improved coupling reagent, monitored under all process, involving Mid-control process to detect MS results|
|25||24 mg||>98%||8.6||48.3768||0.15||Difficult||Semi-automatic synthesis, monitored under all process to ensure coupling efficiency. "O-Acyl isopeptide method" for peptide synthesis|
Example Read Out of NeoPre™. NeoPre™ will analyze individual peptide sequence and assign numerical difficulty values for aggregation, isoelectric point (PI), and hydrophobicity before determining synthetic difficulty and methodology.
|Total||Delivered||Successful Rate||Avg TAT (Days)|
|Difficult||1098||1030||93.81%||36% ≤ 6 days|
|Easy||1169||1128||96.49%||39% ≤ 6 days|
Success Rate of Peptide Synthesis Using NeoPre™. GenScript has a 95% success rate for neoantigen peptide synthesis utilizing our NeoPre™ bioinformatics tool. Overall, we were able to synthesize 94% of predicted difficult peptides.
GenScript offers two synthetic technologies specific for hydrophobic peptide production on top of our standard five synthetic methodologies. One of them is our Hi-Syn technology which utilizes an optimized reaction temperature and condensation reagents to increase coupling efficiency.
Results Using GenScript’s HiSyn Technology
|Project-YTD||Attempted||Synthesized||Synthesis success rate|
Increasing The Success Rate of Difficult Neoantigen Peptides Using HiSyn. GenScript has an overall success rate for difficult neoantigen peptide synthesis of 95% using our technical advances, including our HiSyn technology. Utilizing advanced coupling methods, Hi Syn is a manual technology which has allowed GenScript to synthesize 86.21% of previously un-producible peptides!
|HiSyn||Attempted||Synthesized||Synthesis success rate|
Increasing The Success Rate of Solely Hydrophobic Peptides Using HiSyn. Neoantigen peptides tend to be highly hydrophobic, which negatively affects their silubiloty and therefor their ability to be synthesized. In order to increase the overall success rate of difficult hydrophobic peptides, GenScript’s HiSyn technology is able to synthesize 91% of previously un-producible hydrophobic peptides.
Normal Manual Process
Increasing Purity of Peptides Using HiSyn. The same peptide was unable to be synthesized at 50 mg at 95% purity using our standard manual and automatic technologies, however, utilizing HiSyn, we were able to not only produce the peptide at the required yield, but also at the requested purity as shown through the comparing HPLC chromatograms.
GenScripts double coupling technology uses coupling reagents from our hisyn technology in optimized reaction conditions for our microwave synthesis method. GenScript is the first CRO in the field who can apply such optimized reaction technology utilizing microwave technology.
Results Using GenScript’s Double Coupling Technology
|Project||Total||IPC pass||Synthesis Success rate|
Increasing The Success Rate of Difficult Peptides Using Double Coupling. GenScript has an overall success rate for difficult neoantigen peptide synthesis of 95% using our technical advances, including our HiSyn technology. Utilizing advanced coupling methods, Double Coupling is an automatic technology which has allowed GenScript to synthesize 88.83% of previously un-producible peptides!
Normal Automated Process
Double Coupling Automated Process
Increasing Purity of Peptides Using Double Coupling. The same peptide was unable to be synthesized at 19 mg at 90% purity using our standard manual and automatic technologies, however, utilizing HiSyn, we were able to not only produce the peptide at the required yield, but also at the requested purity as shown through the comparing HPLC chromatograms.
Rather then relying on traditional peptide purification protocols, GenScript has optimized standard methods to enhance peptide purity. Through relying on our expertise in purifying both peptides and proteins of different intrinsic properties, we have designed an enhanced purification protocol utilizing numerous different reagents and purification columns. This allows us to reach peptide purities of even the most difficult neoantigen peptides of up to 98%.
Results Using GenScript’s Optimized Purification Technology
|Project||Total||IPC pass||Synthesis Success rate|
Increasing The Purification of Difficult Peptides Using GenScript’s Optimized Purification technology. Utilizing advanced purification methods, GenScript’s optimized purification methodology has allowed GenScript to purify 86.6% of previously produced peptides which were not able to be synthesized at desired purity.
Optimized Purification Process
Increasing Purity of Peptides Using Optimized technology. The same peptide was unable to be purified at 19 mg at 98% purity using our standard manual and automatic technologies, however, utilizing HiSyn, we were able to not only produce the peptide at the required yield, but also at the requested purity as shown through the comparing HPLC chromatograms.