Affinity Maturation Affinity Maturation

Affinity Maturation

Antibody Optimization
  • Proprietary platform FCMES-AM™
  • Non-Biased Site Saturation Mutagenesis in CDR Regions
  • Guaranteed at least 5-fold Affinity Improvement


Affinity maturation is used to improve antibody affinity and binding interactions to target antigens. This is done naturally in vivo by somatic hypermutation and clonal selection in mammalian B cells, but it can also be done in the lab in vitro by mutagenesis and selection for therapeutic applications.

FCMES-AM™ (Full Coverage Mammalian Expression System for Affinity Maturation) is Biointron's proprietary platform for affinity maturation. Each amino acid in the CDR region will be mutated to the other 17 amino acids (except Cysteine and Methionine) with equal ratios. The clones will be expressed in a mammalian system, and an ELISA binding assay will be conducted as an initial test. This will be followed by SPR (surface plasmon resonance) or FACS (fluorescence-activated cell sorting) for further affinity measurements.

Feel free to reach out to our expert team to explore how Biointron can improve your antibody’s affinity for a better performance.

Affinity Maturation Overview


Non-biased Site Saturation Mutagenesis

  • Full coverage (all CDR regions)
  • Same frequency of occurence for the other 17 amino acids

High Efficiency Screening

  • Guaranteed at least 5-Fold Improvement
  • Affinity ranking ELISA/SPR/FACS

Fast Turnaround

  • 6-8 Weeks
  • HTP Expression in Mammalian System

Working Flowchart

Site-directed saturated mutation
Site-directed saturated mutation
High-throughput mammalian cell expression
High-throughput mammalian cell expression
ELISA and sequencing to identify hot spots
ELISA and sequencing to identify hot spots
Combinatorial mutation design
Combinatorial mutation design

Affinity Maturation
Service Details

Milestone Service Description Deliverables
Sequence Design
  • No-bias Site Saturation Mutagenesis in CDR Region (all 17 amino acid variants for each amino acid in CDR region )
Hot Spots Identification
  • High Throughput Expression in Mammalian System (FCMES-AM™)
  • ELISA Detection
  • ELISA Results
Hot Spots Combination
  • High Throughput Expression in Mammalian System (FCMES-AM™)
  • ELISA Detection
  • ELISA Detection
Antibody Expression & Affinity Detection
  • Antibody Expression & Affinity Purification
  • Affinity Detection by Biacore Full KD or FACS
  • Purified Antibody
  • Plasmid Sequencing Resultsy
  • Affinity Detection Results

Case Study

  • Case 1:

    The parental antibody is VHH, after affinity maturation, off of the VHH improves and the affinity increases 23-fold, 19-fold and 6-fold separately.

    Affinity Maturation Case 1
    Koff of the VHH improves and the affinity increases 6-fold, 23-fold and 19-fold separately.
  • Case 2:

    The parental antibody is mouse IgG, after affinity maturation, the affinity increases 120-fold, 48-fold and 46-fold separately.

    Affinity Maturation Case 2
    Affinity increases 120-fold, 48-fold and 46-fold separately.
“We have our own affinity maturation platform, called FCMES-AM™. I believe this and the research my team is dedicated to developing, is what makes our service the best in the industry. With a guaranteed affinity improvement, you can trust we will get the job done.”
Sufeng Cai
Sufeng Cai
Affinity Maturation Team


  • When is affinity maturation needed?

    Affinity maturation is widely used to improve antibody activity through mutagenesis and selection for downstream applications such as therapeutics and diagnostics. This is often necessary to ensure the affinity towards the target and that the therapeutic potential of antibodies is reached. Optimization can also involve adjusting functionality, cross-reactivity, stability, immunogenicity, and any chemical liabilities.1

  • What is the difference between in vivo and in vitro affinity maturation?

    In vivo

    When a host is repeatedly exposed to the same antigen, the antibodies produced by their immune response will have increasingly greater affinity, avidity, and anti-pathogen activity. This occurs due to somatic hypermutation in the variable CDRs (complementarity-determining regions) of immunoglobulin genes in B cells, and takes place in germinal centers (structures within secondary lymphoid tissues). Clonal selection takes place when follicular dendritic cells of the germinal centers present antigens to the B cells, with only the most competitive B cells surviving to stably conjugate with follicular B helper T cells. After several rounds of selection, high-affinity antibodies will be produced.2

    In vitro

    Similar to the in vivo process, in vitro affinity maturation is used to optimize antibodies, antibody fragments, or other peptides by diversifying the antibody base sequence and isolating higher-affinity binders.

  • What is the principle behind affinity maturation?

    Although antibodies can be generated against countless distinct epitopes, your genome does not have the capacity to hold all the information. Thus, B-cells employ somatic hypermutation, which involves mutations of the genes which encode the antigen-binding region in direct response to the antigen stimulus. Through successive generations and exposure, only B cells producing antibodies that recognize the antigen with high affinity will survive.3

Affinity Maturation Project Commitment

Project Commitment

You will have a dedicated project manager for each project when you work with us, guaranteeing efficient planning, clear communication, and a personalized approach with a strong focus on you. We aim to build trust in all our collaborations, with strong relationships with more than 1,500 biotechnology and pharmaceutical companies.


  • Chan, T. Y., & Groves, A. T. (2021). Affinity maturation: Highlights in the application of in vitro strategies for the directed evolution of antibodies. Emerging Topics in Life Sciences, 5(5), 601-608.
  • Doria-Rose, N. A., & Joyce, M. G. (2015). Strategies to guide the antibody affinity maturation process. Current Opinion in Virology, 11, 137.
  • Alberts B, Johnson A, Lewis J, et al. (2002). The Generation of Antibody Diversity. Molecular Biology of the Cell. 4th edition. New York: Garland Science.

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