Antibody Humanization Antibody Humanization

Antibody Humanization

Antibody Optimization
  • Guaranteed Affinity Comparable to Parental Antibody
  • Quick Turnaround Time of 3-4 Weeks
  • Suitable for IgG, scFv, Fab, VHH, and more


Recombinant antibody technologies have witnessed remarkable advancements and demonstrated significant clinical success. However, a major challenge associated with the use of murine monoclonal antibodies as a therapeutic is their immunogenicity causing a human anti-mouse antibody (HAMA) response. This response occurs when humans react to the foreign protein, causing complications from immune complex formations and resulting in the antibody treatment being ineffective. To tackle this issue, chimeric antibodies were created using genetic engineering techniques, combining human constant domains with mouse variable domains that recognized the human antigen of interest, thus preserving antibody specificity.1

Antibody Humanization Overview

Antibody humanization was further developed by modifying the mouse antibodies to be more similar to human antibodies by CDR (complementarity determining region) grafting. CDRs of non-human antibodies are grafted to human frameworks of high homology. Typically, some vernier zone residues (murine residues in framework regions) are also retained as they have been shown to affect CDR loop conformation and antibody affinity.2 Ultimately, humanized antibodies carry a lower risk for inducing immune responses in humans than mouse or chimeric antibodies.3

Antibody Humanization Overview


Guaranteed Affinity

  • We offer guaranteed affinity (drop less than 3 folds) compared to the parental antibody
  • Less than 5 back mutations for VH/VL each and 7 for VHH
  • If the specified affinity is not attained, there will be no cost

Quick Turnaround Time

  • We deliver humanized antibodies in just 3-4 weeks
  • 10+ years of experience and expertise in humanized antibody design
  • Successful production of more than 20,000 antibodies

Optimal Functionality

  • Receive an affinity detection report with Biacore for a full KD
  • We can validate your humanized antibodies with FACS (fluorescence-activated cell sorting) flow cytometry

Working Flowchart

Gene Synthesis

Plasmid Construction and Preparation

Transient Expression

Affinity Purification

Stringent Quality Control

Affinity Ranking

Antibody Humanization
Service Details

Service Step Service Description Timeline Deliverables
Design of humanized antibody
  • Bioinformatics analysis
  • 3D structure modeling and identification of back mutations
  • Human germlines selection
  • In silico CDR-grafting
  • Sequence optimization
2-3 days
  • Designed humanized antibody sequences
Humanized antibody recombinant production variants 9 to 25
  • Codon optimization and gene synthesis
  • Subcloning into expression vector
  • Transient expression
  • Purification
  • QC analysis
2 weeks
  • Purified humanized antibody samples for in house test
Affinity Ranking
  • Affinity Determination with soluble antigen via Biacore 8K
1 week
  • Detailed report

Case Study

  • Case 1:

    The affinity of humanized antibody (measured by SPR, Biacore 8K) is comparable with the parental antibody.

    Antibody A
    Antibody A
    Humanized Antibody A
    Humanized Antibody A
    The affinity of humanized antibody is comparable with the parental antibody.
  • Case 2:

    The parental antibody is VHH. Based on the SPR results, the affinity of humanized antibodies is similar to that of the parental antibody.

    Antibody Humanization Case 2
"As a scientist at Biointron, my team’s collaborative spirit and shared commitment to excellence creates a positive and dynamic environment where I am encouraged to work to the best of my ability. Combined with cutting-edge technologies, you can be confident in the quality of our antibody humanization service.”
Jiwen Du
Jiwen Du
Antibody Optimization Team


  • What is CDR grafting?

    Complementarity Determining Regions (CDRs) are the antigen-binding regions of an antibody, and they play a crucial role in determining the antibody's specificity and affinity for its target antigen. In CDR grafting, the CDRs of a non-human antibody are transferred or grafted onto a human antibody framework. This process involves molecular modeling and structural analysis to ensure that the grafted CDRs fit properly into the human framework.

  • Why are back mutations needed?

    CDR grafting can lead to a partial or complete reduction in affinity. To counter this effect, certain amino acid residues from the murine framework sequences must be preserved and reintroduced at the corresponding humanized antibody sequence positions. Back mutations are performed iteratively through computational modeling and experimental validation to fine-tune the humanized antibody, ensuring that it retains its target specificity and functional properties while minimizing its immunogenic potential.

  • What applications are there for antibody humanization?

    Antibody humanization made it possible to use antibody therapeutics directed against diseases that require long-term treatment, such as cancer and autoimmune diseases. For example:

    • Pembrolizumab: mAb cancer immunotherapy to treat melanoma, lung cancer, head and neck cancer, Hodgkin’s lymphoma, and stomach cancer.
    • Ibalizumab: IgG4 mAb HIV treatment as a CD4 domain 2-directed post-attachment HIV-1 inhibitor, for adult patients who were previously treated and are resistant to other available therapies.4


  • Pornnoppadol, G., Zhang, B., Desai, A. A., Berardi, A., Remmer, H. A., Tessier, P. M., & Greineder, C. F. (2021). A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain. PLOS ONE, 16(10), e0252558.
  • Safdari, Y., Farajnia, S. (2013). Antibody humanization methods – a review and update. Biotechnology and Genetic Engineering Reviews, 29(2), pp. 175–186.
  • Harding, F. A., Stickler, M. M., Razo, J., & DuBridge, R. B. (2010). The immunogenicity of humanized and fully human antibodies: Residual immunogenicity resides in the CDR regions. MAbs, 2(3), 256-265.
  • Lu, RM., Hwang, YC., Liu, IJ. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020).

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