Home » Biologics Discovery » Antibody Engineering » Antibody Affinity Maturation

Service Introduction

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.

Construction of combinatorial library & screening
Production and characterization of affinity-matured antibody

MOE Homology Modeling Platform

  • 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.

Precise Mutagenesis Library (PML) Platform

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

PML NNK library
Coverage rate High Low
Primers by Chip Oligo degenerate primers
Type of codon customized random
Type of amino acids customized random
Unexpected codon ×
Codon optimization ×

FASEBA (Fast Screening for Expression, Biophysical-properties and Affinity) High-throughput Screening Platform

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

  • FA   Fast

    S     Screening of

    E     Expression

    B     Biophysical-properties and

    A     Affinity

Service Package

Description Deliverable Timeline
MOE Homology Modeling
  • All the optimized sequences
  • 3 Purified antibodies with the best affinity, 0.5mg/antibody
  • Guarantee at least 1 sequence has 5-10 fold of affinity improvement
8-13 weeks
PML library construction & FASEBA screening
Construction of combinatorial library & screening
Production and characterization of affinity-matured antibody (Up to three)

For international shipping, please refer to the time provided by PM during project discussion.

Case Study

Ligand Analyte Chi² (RU²) ka (1/Ms) kd (1/s) KD (M) Rmax (RU)
Target B WT 7.96E-01 1.40E+05 1.16E-04 8.29E-10 64.7
Target B variant 1 7.60E-01 1.97E+05 3.73E-05 1.90E-10 38.8
Target B variant 2 1.23E+00 1.97E+05 1.00E-06 5.08E-12 51.3
Target B variant 3 4.36E-01 1.52E+05 1.67E-05 1.09E-10 39.7
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Related Services

GenScript ProBio offers our customers a one-stop solution for all outsourcing needs, providing comprehensive upstream and downstream services of monoclonal antibody sequencing service.

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