Overview

At GenScript, we have the technical means & experience to synthesize large numbers of challenging neoantigen peptides and provide tailored optimization tools for your downstream applications.

Stages of Neoantigen Precision Immunotherapy

GenScript’s Neoantigen Peptide Synthesis Service can be used for everything from functional screening of neoantigen candidates for TCR therapy to clinical stage vaccine immunogenicity testing.

Stages of Neoantigen Precision Immunotherapy

What Makes Neoantigen Peptides so Difficult to Produce?

Characteristic Description Difficulty
Length The length 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?

Sequence Analysis

Sequence Analysis

Peptide Synthesis

Peptide Synthesis

Peptide Purification

Peptide Purification

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.

Read the full case study here!

Length Yield Purity pI Aggregation Hydrophobicity Prediction Recommended Protocol
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
25 4 mg >98% 6.4 39.3832 0.05 Difficult R&D

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)
Total 2267 2158 95.15%
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
Total 232 200 86.21%

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
Hydrophobic peptide 88 80 91%

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

Peptide Synthesis Normal Manual Process

HiSyn Process

Peptide Synthesis HiSyn 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
Double coupling 385 342 88.83%

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

Peptide Synthesis Normal Automated Process

Double Coupling Automated Process

Peptide Synthesis 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
Purification 67 58 86.6%

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.

Normal Process

Peptide Purification Normal Process

Optimized Purification Process

Peptide Purification 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.

Tailored Synthesis For Any Application!

TCR-Antigen Affinity &
Immunogenicity
Screening
TCR-Antigen Affinity & Immunogenicity Screening
Personalized Cancer
Vaccine (PCV)
Personalized Cancer Vaccine (PCV)
PCV Efficacy
Screening
PCV Efficacy Screening

Features of GenScript’s Service

Trustworthy Partner Trustworthy Partner
in Neoantigen Synthesis
High Success Rate High Success Rate for
Synthesis of Difficult Peptides
Service Flexibility Service Flexibility for
Personalized Therapy

"Reliable Reputation Amongst the Neoantigen Community"

"Screening hundreds of neoantigens in mice to validate the generalizability of SNP-7/8a was a tremendous undertaking that could not have been possible without the diligent efforts of GenScript's peptide chemists who were able to rapidly synthesize even the most challenging sequences at the high purity required."

―Geoffrey Lynn, Ph.D.
Avidea Technologies' CEO

Proven Reliability

Strong Application Expertise icon

Strong Application Expertise

Produced 1000’s of peptides for TCR therapy screening for 3T Biosciences

Synthesized Difficult peptides at varying amounts for Avidea’s novel SNAP PCV delivery system

Generated highly immunogenic peptides for DNA-based PCV efficacy screening

Track Record of Success icon

Track Record of Success

15 years of peptide synthesis production with a 98% success rate

Successful production of neoantigen peptides for the Top 50 immunotherapy companies and academic institutes

Continuous Communication icon

Continuous Communication

Global presence allowing for 24/7 communication and technical support

Project-specific managers and milestones

"Utilizing Reliable Synthesis Technologies & Tools for Superior Immunogenicity"

In 2019 GenScript manufactured 2500 neaontigen peptides with a 95% success rate using our tailored neoantigen peptide synthesis platform.

How Do We Do It?

checklist icon

Step One: Input EVERY one of your peptides through our proprietary NeoPre™ bioinformatics algorithm to determine synthesis difficulty

Do you want to Know More About GenScript’s Neopre Bioinformatics Algorithm? Check out our Webpage for technologies for Difficult Neoantigen peptide Synthesis Here!

Peptide # Sequence Length Aggregation Hydrophobicity pI Synthetic Difficulty
Example 1 XXXXXXXX 25 4.18 -0.032 11.83 Middle
Example 1 XXXXXXXX 25 4.18 -0.032 11.83 Middle
Example 2 XXXXXXXX 25 31.63 0.49 10.06 Hard
Example 3 XXXXXXXX 25 -4.77 -0.58 6.78 Simple
pick the right synthesis platform icon

Step Two: Based on difficulty and project details we help you pick the right synthesis platform to maximize success

Do you want to Know More About GenScript’s Unique Synthetic Methodologies for Difficult Neoantigen Peptide Synthesis and Purification? Check out our Webpage Here!

Technology Use Case
Microwave Automated, High Yield, Fast TAT
Liquid-Phase For modified peptides
Solid-Phase Higher purity peptides
library design tools icon

Step Three: Once your peptides have been produced, GenScript offers library design tools to assist you optimize any neoantigen sequence

Library Use
Alanine Scanning Determine the most important individual amino acids
Overlapping Discover the perfect peptide length and sequence
T-Cell Truncation Identify the best epitope for a specific activity

"Reliable Peptide Yields In Your Timeline"

Neoantigens may require different yields, TAT, number, purity, ect, depending on their downstream application. To accommodate the needs of the neoantigen community, ALL of GenScript’s neoantigen peptide services are fully flexible and customizable.

Example Project 1: TCR Affinity and Immunogenicity Screening

GenScript offers a wide range of services to fit the needs of customers working on the beginning stages of T-cell therapy to clinical stage efficacy screening. Learn the various ways GenScript’s neoantigen peptide synthesis service can be customized to fit the peptide requirements of each phase, even including TCR affinity maturation and epitope identification! Read More »

Project Goal: Generate an Allogenic T-cell Therapy for Patient with Squamous Cell Carcinoma

Phase I: Phase I: After tumor biopsy and NGS, 300 different neoantigens were identified as possible candidates. These sequences were sent to GenScript to synthesize as a crude peptide library to be used for in-vitro functionality screening of TCR Binding Affinity and Efficacy.

# Peptides Analyzed # Peptides Synthesized Avg Peptide length Yield Purity TAT (B.D) Quality Control Synthesis Methods Used Delivery
300 290 30 AA 2 mg/peptide 70% 20 RP-HPLC
LC-MS
Simple: 68% of Peptides Synthesized Using Microwave Technology
Difficult: 22% of Peptides Synthesized Using LP/SP Manual technology
Lyophilized Peptides in Individual Aliquot Tubes

Phase II: In-vitro functionality screening identified a top candidate, however, in order to increase the binding affinity and efficacy of the TCR, researchers decided to design and generate an overlapping library to map the specific interaction between neoantigen epitope and TCR.

Library Type Length Offset Overlap # of Peptides Purity Yield Delivery Format
Overlapping 11 AA 3 AA 6 AA 100 80% 0.5 mg/peptide Pooling all quantity of partial peptides (20 peptides/pool)

Phase III: Once the specific epitope was identified, researchers designed and generated an alanine scanning library to determine which amino acids were most important for therapeutic efficacy, prior to affinity maturation of the TCR.

Library Type Length Purity Yield Delivery Format
Alanine Scanning 11 AA 98% 4 mg/peptide Lyophilized Peptides in Individual Aliquot Tubes (1 mg/tube)

Example Project 2: PCV Development

When generating a PCV, researchers require an array of peptide services due the specific needs of in vitro functionality screening, pre-clinical safety basement in laboratory models, and clinical efficacy analysis. Learn how GenScript’s neoantigen peptide synthesis service has flexible services which allow for reliable peptides for any phase of PCV development. Read More »

Project Goal: Generate a Personalized Cancer Vaccine for a patient with Glioblastoma

Phase I: After tumor biopsy and NGS, 150 different neoantigens were identified as possible candidates. These sequences were sent to GenScript to synthesize as a purified peptide library to be used for in-vitro functionality screening.

# Peptides Analyzed # Peptides Synthesized Avg Peptide length Yield Purity TAT (B.D) Quality Control Synthesis Methods Used Delivery
150 145 28 AA 2 mg/peptide 75% 20 RP-HPLC
LC-MS
Solubility Testing
Simple: 60% of Peptides Synthesized Using Microwave Technology
Difficult: 40% of Peptides Synthesized Using LP/SP Manual technology
Lyophilized Peptides in Individual Aliquot Tubes (1 mg/vial)

Phase II: In-vitro functionality screening identified 20 peptides for pre-clinical PCV development for non-human primate.

# Peptides Analyzed # Peptides Synthesized Avg Peptide length Yield Purity TAT (B.D) Quality Control Synthesis Methods Used Delivery
20 19 35 AA 500 mg/peptide 98% 30 RP-HPLC
LC-MS
Solubility Testing
Simple: 30% of Peptides Synthesized Using Microwave Technology
Difficult: 70% of Peptides Synthesized Using LP/SP Manual technology
Lyophilized Peptides in Individual Aliquot Tube

Phase III: After the vaccine was deemed “safe” through pre-clinical testing; the vaccine was used in a human patient. After inoculation, researchers required “vaccine” and “non-vaccine control” neoantigen peptides for clinical in-vitro efficacy screening of patient derived PBMC’s.

# Peptides Analyzed # Peptides Synthesized Avg Peptide length Yield Purity TAT (B.D) Quality Control Synthesis Methods Used Delivery
25 25 34 AA 14 mg/peptide 70% 20 RP-HPLC
LC-MS
Simple: 35% of Peptides Synthesized Using Microwave Technology
Difficult: 65% of Peptides Synthesized Using LP/SP Manual technology
Lyophilized Peptides in Individual Aliquot Tube (2 mg/vial)

Check Out GenScript’s Neoantigen Peptide Resources

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