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.
Why choose ProteoGenix for your antibody-drug
conjugate development project?
Recognized antibody humanization experts
Get access to experts having more than 25 years of experience and unrivalled track record in antibody humanization.
3D model building
Our 3D model building platform guarantees biological function conservation.
IP free
You get the full ownership of the humanized antibodies.
Full set of characterization services
ELISA, KD determination against soluble antigen or cells, thermostability, aggregation rate, IC50…
XtenCHOTM
Benefit from the most productive transient expression system to produce all your humanized monoclonal antibody variants!
Format diversity
We optimize sequence or humanize all kinds of antibody formats: full length IgG, scFv, Fab, VHH (nanobodies)…
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
Our monoclonal antibody humanization service content
| Step | Content | Timeline | Deliverables |
|---|---|---|---|
| Parental antibody sequencing from hybridoma |
|
~2-3 weeks |
|
| Chimeric antibody expression and purification |
|
~7-9 weeks |
|
| Design of humanized antibody |
|
~2 weeks |
|
| Transient recombinant production of 9 to 18 humanized antibody variants |
|
~7-9 weeks |
|
| Characterization of humanized monoclonal antibody variants |
|
To be determined |
|
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”.
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.