We understand that antibody humanization is one of the most critical steps on your way to IND. This is why our experts are some of the world’s most renowned scientists in the field, boasting an impressive track record. We currently have 3 therapeutic antibodies already on the market and more than 30 in preclinical and clinical stages. We consider each new monoclonal antibody humanization as a unique and exciting challenge in bringing your compound to the clinic.

Our monoclonal antibody humanization process

Antibody sequencing starting from hybridoma cell line

  • RNA extraction and purification
  • Reverse Transciption
  • PCR amplification
  • Sequencing

Chimeric antibody bioactivity evaluation

  • Gene synthesis including codon optimization
  • Subcloning in an expression vector
  • Chimeric antibody production and purification
  • QC analysis

Humanized monoclonal antibody design

  • Identification of back mutations by 3D model building
  • Selection of the most relevant human germlines
  • In silico CDR grafting and sequence optimization

Humanized antibody production (9 to 18 variants)

  • Gene synthesis including codon optimization
  • Subcloning in an expression vector
  • Transient transfection, expression and purification
  • QC analysis

Characterization of the humanized variants

  • ELISA against antigen
  • Antibody affinity against soluble antigen or cell
  • Aggregation rate
  • Thermostability
  • Glycosylation profile
  • Endotoxin detection
Step Content Timeline Deliverables
Parental antibody sequencing from hybridoma
  • Hybridoma cell line provided by customer or developed by ProteoGenix
  • RNA extraction and purification
  • Reverse transcription
  • cDNA amplification
  • Sequencing analysis
~2-3 weeks
  • Detailed report
  • Antibody sequence
    Chimeric antibody expression and purification
    • Codon optimization for Mammalian expression system
    • Gene synthesis
    • Subcloning into expression vectors
    • Transient expression
    • Purification
    • QC analysis: SDS-PAGE, WB, UV280
    ~7-9 weeks
    • Detailed report
    • Chimeric antibody sample for testing
    Design of humanized antibody
    • Bioinformatics analysis
    • 3D structure model building and identification of back mutations
    • Human germlines selection
    • In silico CDR-grafting
    • Sequence optimization
    ~2 weeks
    • Detailed report
    • Chimeric antibody sample for testing
    Transient recombinant production of 9 to 18 humanized antibody variants
    • Codon optimization for Mammalian expression system
    • Gene synthesis
    • Subcloning into expression vectors
    • Transient expression
    • Purification
    • QC analysis: SDS-PAGE, WB, UV280
    ~7-9 weeks
    • Detailed report
    • Discussion with one of our therapeutic antibody expert
    Characterization of humanized monoclonal antibody variants
    • Affinity analysis by ELISA against the antigen
    • Analysis of antibody aggregates (SEC-HPLC)
    • Affinity Determination (Kd) against soluble antigen (SPR/Biacore X100)
    • Affinity Determination (Kd) against antigen expressed on cell surface (SPRi/ Horiba XelPlex)
    To be determined
    • Detailed report
    • Discussion with one of our therapeutic antibody expert

    Options available:

    • DSC Analysis (Thermostability Analysis by Differential Scanning Calorimetry)
    • Glycosylation profile (LC/MS)
    • Endotoxin detection test
    • FACS analysis

    Which factors should be considered in monoclonal antibody humanization?

    The main objective of the antibody humanization process is to develop an antibody sequence compliant with World Health Organization guidelines (WHO) for humanized antibody. Basically, an antibody is considered as humanized when the sequence identity between the antibody and a human germline sequence (from the IMGT database) is of at least 85%. The difference between humanized and fully human antibody depends on the antibody origin; antibodies originating from non-human species will be classified as humanized after humanization whereas sequences derived from human will be considered as “fully human antibodies”.

    Monoclonal antibody humanization

    Reaching this 85% goal means modifying the antibody sequence in the variable regions. Thus, this antibody engineering work can have a critical impact on the physicochemical and pharmacological properties of the final product and should be performed carefully. For this reason, ProteoGenix combines its unrivalled know-how in antibody engineering with experts boasting more than 25 years of experience and a strong track record in antibody humanization. Our humanization process encompasses CDR grafting, 3D molecular model building and sequence optimization.

    3D molecular model building is an important part of this process as it allows the analysis of the contributions of individual amino-acids localized in the murine CDR loops and in the framework regions. This step is essential for identifying residues eligible for back mutations and those which can be mutated for further properties optimization. Amino-acids involved in the variable regions can be classified as follows:

    • Residues involved in the antibody-antigen interaction,
    • Residues playing a structural role, for instance by maintaining the CDR loop conformation or stabilizing the VH-VL interaction,
    • Residues interacting with the solvent.


    Residues from the murine framework regions considered as critical to maintain the conformation of the CDR loops and the antibody bioactivity will be back mutated in the selected human germlines.

    The human germline selection is based on sequence homology, but other humanization variants, demonstrating lower sequence homology but presenting good physicochemical properties, can be tested. In a humanization project, ProteoGenix selects several heavy chains and light chains in order to generate between 9 and 18 combinations.

    The different antibodies generated are further characterized and compared to the reference parental and/or chimeric antibody in order to assess their:

    • Immunogenicity
    • Physicochemical properties (stability, aggregation rate…),
    • Pharmacological properties (affinity, specificity…),
    • Manufacturability.


    Following successful testing, lead candidates can be selected for further humanization and/or CDRs sequence optimization for improvement of biophysical properties (heterogeneity, fragmentation, aggregation…) and manufacturability bioproduction.

    Comprehensive antibody affinity maturation services can be done via phage display using custom libraries generated by random or target mutagenesis.

    How are mouse monoclonal antibodies humanized: current approaches and limitations

    Mouse monoclonal antibodies were the first to ever be produced in vitro. However, early-stage clinical evaluation of these molecules led to the quick discovery of their undesirable side-effects. These side-effects consisted of a moderate allergic response that became known as the human anti-mouse antibody (HAMA) response. Its discovery prompted the development of antibody humanization techniques that have become indispensable for the development of therapeutic antibodies from nonhuman hosts.

    Are humanized antibodies still relevant for therapeutic applications?

    As of February 2020, 82 therapeutic antibodies were granted approval by the FDA or EMA for clinical use in the US or EU, respectively. Of these, 39 are humanized immunoglobulins generated in mice or rabbits. This data reflects the fruits of the past decade of research in antibody humanization. Moreover, it shows that humanized antibodies still stand on equal terms with fully human antibodies when it comes to rates of clinical approval.
    Over the years, scientists developed different techniques to accomplish antibody humanization. Interestingly, many of these techniques seem to apply to antibodies independently of their original host. For this reason, as antibodies from species like rabbit and camelids become increasingly relevant, so will the use of techniques that aim at humanizing these biomolecules.

    Underlying principles of monoclonal antibody humanization

    Immunoglobulins consist of four peptide chains – two identical heavy chains (H) and two identical light chains (L). Each chain contains a variable (V) and a constant domain (C), responsible for interacting with the antigen or with other key elements of the immune system, respectively. Within each V domain (VL and VH) there are three non-consecutive complementarity-determining regions (CDRs) loops or hypervariable regions, that directly interact with an antigen.
    For this reason, CDRs and adjoint regions contain the most important information regarding antigen-specificity in a given molecule. This knowledge has led to the development of one of the most popular methods of antibody humanization – CDR-grafting – which takes these regions and integrates them into the framework of a human antibody. Thus, reducing the risk of the molecule being recognized as foreign by the patients’ immune system.
    Most antibody humanization techniques start by generating a chimeric IgG-based antibody containing the V region of the xenogeneic molecule and the C region of a typical human IgG molecule. After the expression of a stable chimeric molecule, humanization efforts focus on the V region itself through different approaches.

    Underlying principles of monoclonal antibody humanization


    To this day, CDR-grafting continues to be the preferred method for antibody humanization. This technique implies the use of a “donor” antibody containing the antigen-specific CDRs and the “acceptor” antibody that will serve as the framework for the hybrid molecule. The key step of this technique is thus the choice of an adequate “acceptor” molecule.

    Typically, human frameworks with the highest homology to the same regions of the xenogeneic antibody are chosen as “acceptor” molecules.

    Interestingly, early attempts at humanizing mouse antibodies through the CDR-grafting approach resulted in a significant loss of affinity and specificity. In many cases, this loss resulted from inaccurate annotation of the CDRs sequences, inappropriate choice of human frameworks, and erroneous identification of homologous regions between the “donor” and “acceptor” molecule.

    However, it soon became evident that grafting the CDR residues alone was insufficient to guarantee the binding efficiency, because adjacent residues could also affect the conformation of the antigen-binding site. These key regions in the vicinity of the CDR loops became known as “Vernier zone residues”. They were shown to affect the stability, flexibility, and structure of the CDR loop, and thus, impact the affinity and specificity of the molecule. For this reason, current CDR-grafting strategies now include the grafting of these complementary regions as well.


    Interestingly, other authors have taken a completely different approach on how to match “acceptor” and “donor” antibodies. Instead of focusing in the homology between the framework regions (FWR) of the human and xenogeneic molecule, some researchers are focusing on matching these molecules based on the homology of the CDR regions. Why? Because of the importance of adjacent residues to the conformation of an antibody.

    Unlike the conventional CDR-grafting approach, humanization by CDR homology does not attempt to preserve the residues from the Vernier zone, instead, it tries to find a pre-existing human IgG antibody with comparable structural organization. Antibodies generated by this method were found to retain a significantly higher affinity than antibodies generated via the framework region homology method.

    These results suggest this method is a suitable alternative to the current approach at CDR-grafting humanization. However, to the best of our knowledge this approach has not become as widespread as the former in therapeutic antibody development. Possibly because CDR-grafting by FWR homology was developed earlier than this alternative approach.


    Antibodies humanized by the CDR-grafting technique may still elicit an immune response in patient. To minimize this response, the grafted regions may be further restricted to the specificity-determining residues (SDRs).

    SDRs are the most important residues involved in antibody-antigen interaction within the CDRs. By grafting these residues alone, instead of grafting the entire CDR, researchers aim at reducing the anti-idiotypic (anti-Id) response. Several researchers have tested this approach in murine antibodies and concluded that these molecules had a binding affinity comparable to their CDR-grafted counterparts and showed a lower immunogenicity in patients.

    SDR-grafting follows the same principles as conventional CDR-grafting by FWR homology in terms of choice of appropriate human frameworks and correct annotation of the CDR region. However, little work has been published recently on this topic, suggesting that SDR-grafting is not a widely used technique for antibody humanization.


    The variable domain resurfacing approach to antibody humanization emerged in the early 1990s. Unlike SDR and CDR-grafting, the resurfacing method takes a more holistic approach to antibody humanization.

    Instead of focusing on specific and well-defined regions, resurfacing seeks to identify surface framework residues that can potentially cause an immune response in patients, and replace them by the most common residues found in human antibodies in those same positions. In other words, resurfacing implies the replacement of problematic residues in the FWR surface of the xenogeneic molecule, by safer residues typically found in human antibodies.

    Several studies show that antibodies humanized by this approach had a longer half-life, reduced immunogenicity, and comparable in vitro activity. However, very few antibodies have actually been humanized using this approach, suggesting there is a need to gather more data to understand the real potential to this approach at humanization.

    Antibody germline-based humanization

    Of all the current approaches to antibody humanization, germline-based humanization is perhaps the most innovative and controversial one. It is based on the knowledge that human germline frameworks are better tolerated by the immune system than framework sequences from fully mature IgG antibodies. Unlike germline sequences (pre-affinity maturation), the framework of IgG antibodies undergoes many somatic hypermutations during the affinity maturation process. For this reason, even fully human IgG molecules are known to cause an allergic reaction, known as the human anti-human antibody (HAHA) response.

    Despite some antibodies have been humanized through this approach and still managed to retain their therapeutic effectiveness, there also has been some studies reporting the opposite effect. For this reason, more research is necessary to understand the true potential of this approach.