Antibody Humanization Development Platform
Humanized antibodies trigger minimal or no response from the human immune system. According to Cognitive Market Research, the global Antibody Humanization market size is USD 92.5 million in 2024 and will expand at a compound annual growth rate (CAGR) of 13.00% from 2024 to 2031. Alpha Lifetech has a mature antibody humanization platform, with a success rate of over 98% for humanized antibodies. We have multiple humanization strategies, including CDR grafting, SDR grafting, chain shuffling, phage display, etc., which can be selected according to your experimental needs. We can not only provide the humanization of antibodies from multiple species, such as mice, rabbits, alpacas, camels, etc., but also the humanization of antibodies in various forms, such as scFv, Fab, and nanobodies. We have professional technical personnel to provide antibody production, purification, and validation services, including mouse monoclonal antibody production, chimeric antibody production, humanized antibody expression and purification, and humanized antibody characterization and analysis. With a high success rate, high purity, and low immunogenicity, we guarantee humanized antibody production.
Introduction to Antibody Humanization
Monoclonal antibody therapy can be used for the treatment of autoimmune diseases, cancer, and other diseases. Monoclonal antibodies are produced by immunizing mice or other animals. When used to treat diseases in the later stage of monoclonal antibody production, the immunogenicity of non-human antibodies cannot be ignored. The main principle of humanization is to integrate non-human framework residues, such as hypervariable complementarity-determining regions (CDRs), into the human framework, producing sequences that retain the original antibody characteristics while preventing immunogenicity. Mouse immunization and subsequent mouse sequence humanization remain two of the main pathways for discovering therapeutic antibodies, which mainly go through several stages: mouse antibody production, monoclonal antibody chimerization, chimeric murine human monoclonal antibody, and the development process of fully humanized monoclonal antibody. At present, research mainly includes transgenic mice (transferring human B cell genes into mice), high-throughput screening using yeast or phage display technology, CDR grafting (inserting parental CDRs into human sequences), transplant-specific determination residues (SDR), framework shuffling, and other methods.

Fig 1: Schematic overview of antibody humanization from murine antibodies (green domains) to fully human antibodies.
Antibody humanization strategy
CDR Grafting
CDR grafting is achieved through a mammalian expression system based on recombinant DNA technology, and its main steps are:
(1) Develop corresponding antibodies in mice (or non-human sources), isolate DNA encoding antibodies, and clone them into vectors for sequencing (or directly perform single-cell sequencing).
(2) To determine the DNA sequence corresponding to the antibody CDR and to determine the target binding specificity;
(3) Select the human framework region (FR) to transplant non-human CDRs and construct a new antibody gene.
(4) Perform a three-dimensional analysis of conflicts between non-human CDRs and human FRs to generate recovery mutations to stabilize the loop and prevent loss of affinity or structural integrity of the final humanized antibody.

Fig 2: Schematic overview of the complementarity determining region (CDR) grafting.
Framework shuffling
Framework shuffling relies on phage display technology and phage library screening technology to obtain high-affinity antibodies. Oligonucleotides encoding human antibody heavy chain frameworks are used as templates, CDR regions are encoded as primers, and PCR amplification is performed to denature the PCR product to obtain single-stranded DNA. Single-stranded DNA of VL and VH genes purified with streptavidin is reconstructed onto vectors using T4 DNA ligase and restriction enzymes, incubated with bacteriophages, and amplified by bacteriophages, and a phage display library is constructed. Three rounds of screening with corresponding antigens are performed, and positive clones are finally verified using ELISA and other methods.

Fig 3: Schematic illustration of the complementarity determining region (CDR) grafting and framework (FR) shuffling humanization strategies. (A) The main procedure of CDR grafting. (B) The main process of the FR shuffling. (Reference source: Wang Yongmei, et al., Comparison of “framework shuffling” and “CDR grafting” in the humanization of a PD-1 murine antibody.)
Phage Display
The production of humanized antibodies is based on the method of displaying specific antibodies on the surface of bacteriophages, mainly constructed through human antibody phage libraries. The source of the library is PBMCs isolated from human peripheral blood to construct phage libraries, from which corresponding sequences are screened. This sequence has the complete human sequence, but the produced antibodies are antibody fragments, such as scFv, Fab, and nanobody.
Antibody Humanization Service Workflow
Service Steps | QC Standard | Timeline |
---|---|---|
Antibody Isolation | Purity >95% | 1-2 weeks |
Sequence Analysis | Accurate identification of non-human sequences | 1 week |
CDR Grafting and Framework Grafting | Successful integration and expression | 2-3 weeks |
Expression and Purification | High yield and purity | 2-3 weeks |
Functional Validation | Affinity and specificity assessment | 1 week |
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