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MONOCLONAL ANTIBODY HUMANIZATION SERVICES

Home/Therapeutic antibody development/Monoclonal antibody humanization

We understand that antibody humanization is one of the most critical steps on your way to IND. That's why our experts are part of the world most renowned scientists in this field boasting an impressive track record. 2 therapeutic antibodies on the market and more than 30 in preclinical and clinical stages are some of our key figures. We consider each new humanization project as a unique and exciting challenge to bring your compound to the clinic.

Why choose ProteoGenix for your monoclonal
antibody humanization?

Monoclonal antibody humanization experts

Recognized antibody humanization experts

 Get access to experts having more than 25 years of experience and unrivalled track record in antibody humanization.

3D modelling

Our 3D molecular modeling platform guarantees biological function conservation.

IP free antibody humanization services

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…

Humanized antibody production with XtenCHO

XtenCHOTM

Benefit from the most productive transient expression system to produce all your humanized monoclonal antibody variants!

Humanized antibody formats

Format diversity

We optimize sequence or humanize all kinds of antibody formats: full length IgG, scFv, Fab, VHH (nanobodies)…

Our monoclonal antibody humanization process

Hybridoma sequencing
  • Antibody sequencing starting from hybridoma cell line

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

Chimeric antibody production
  • Chimeric antibody bioactivity evaluation

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

Go/No Go1

Humanized monoclonal antibody engineering
  • Humanized monoclonal antibody design

-Identification of back mutations by 3D molecular modelling
-Selection of the most relevant human germlines
-In silico CDR grafting and sequence optimization

Go/No Go2

Humanized antibody production
  • Humanized antibody production (9 to 18 variants)

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

Go/No Go3

Humanized monoclonal antibody characterization
  • 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
  • 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 modeling and identification of back mutations
  • Human germlines selection
  • In silico CDR-grafting
  • Sequence optimization
~2 weeks
  • Detailed report
  • Discussion with one of our therapeutic antibody expert
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
  • Purified humanized antibody samples for testing
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 the 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%. 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 implies to modify 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 having more than 25 years of experience and a strong track record in antibody humanization. Our humanization process encompasses CDR grafting, molecular modeling and sequence optimization.

Molecular modeling is an important part of this process as it allows analyzing the contributions of individual amino-acids localized in the murine CDR loops and in the framework regions. This step is essential to identify 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 antibody 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.