Guarantee at least 10 fold affinity improvement
Delivery as fast as 8 weeks
Affinity maturation is known to improve the antibody-antigen binding kinetics for drug optimization. Despite the popularity of NNK (degenerate primer, N= A/C/G/T, K= G/T) mutagenesis and phage library, inherent limitations exist, such as unequal mutation distribution, WT sequence enrichment, and limited saturation mutation coverage within the Complementarity-Determining Region (CDR). To address these limitations, GenScript ProBio combined the power of efficient, unbiased mutagenesis by PML (precise mutagenesis library) technology and the high efficiency of the HTP FASEBA (Fast Screening for Expression, Biophysical-properties, and Affinity) affinity screening platform.
80+ projects delivered
Guarantee at least 10-fold affinity improvement
By Dec 2022
During affinity maturation (AM), the theoretical size of saturation mutation covering all CDR is at the level of 20E60, which is difficult to build in display libraries. Instead of confining the region of saturation mutation to very few residues in each CDR, GenScript ProBio employed more powerful tools, including Molecular Operating Environment (MOE) based structure modeling, PML, the high efficiency of the HTP FASEBA affinity screening platform, two-step mutation strategy, and a Surface Plasmon Resonance (SPR) kinetics analytical platform (Biacore 8K) to cover all possible mutations in each CDR.
MOE(Molecular operating environment) is an integrated computer-aided molecular design platform. This software is linked to the RCSB/PDB database and more than a dozen of classical protein family structure databases (e.g., transporter family, GPCR family protein kinases, etc.). Potential residues are identified by structure modeling combined with internal database, which can guide antibody optimization.
PML is a site-saturation mutagenesis library or a saturation scanning mutagenesis library. Each amino acid is mutated into the other 19 amino acids with the same distribution. ProBio will identify 3-5 mutation hot spots by screening a PML. Then based on the hot spots, ProBio will construct a combinatorial mutation library to find the best 3-5 combinations with the desired affinity. This method improves the screening efficiency and increases the probability of obtaining high-affinity sequences.
Generation of saturation mutation library with similar frequency and 100% coverage
PML is created by on-chip synthesized Oligo to achieve controlled distribution of mutation
Sequencing saturation mutation library by NGS to verify distribution of mutation.
Figure. NGS disclosed that each CDR residue was mutated to the other 19 residues (excluding itself) with similar frequency
Comparison with traditional NNK library
FASEBA is a patented E.Coli based system that selects good affinity, expression level, and thermostable candidates. It is used to select the best protein binders from a large number of protein candidates in a high throughput fashion, without actually purifying these proteins.
Each saturation mutation library is transformed into FASEBA E.coli based expression system
S Screening of
B Biophysical-properties and
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