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Service Overviews

Background: In order to overcome the heterologous reaction of murine monoclonal antibody, the framework of IgV region can be humanized based on the chimeric antibody, and the humanized rate can reach more than 90%. Based on this technology, SanyouBio has integrated computer 3D structure simulation and database sequence analysis, and launched the in-depth humanization service of antibodies.


Methods: The in-depth antibody humanization service integrates the technologies of non-human sequence 3D structure simulation and analysis, eukaryotic expression and purification identification, affinity and functional assay, etc. On the premise of retaining both specificity and affinity of the antibody, the antibodies with more than 95% humanization were eventually obtained.


Advantages: Based on computer 3D structure simulation, the antibodies with more than 95% humanized , and back mutations are less than 6.


Cases: Since the launch of the service in 2016, SanyouBio has completed more than 200 cases of humanized antibody design, of which more than 40 designed antibodies are in the clinical application stage, and 7 are in the clinical trial stage.
Service Overviews Background: In order to overcome the heterologous reaction of murine monoclonal antibody, the framework of IgV region can be humanized based on the chimeric antibody, and the humanized rate can reach more than 90%. Based on this technology, SanyouBio has integrated computer 3D structure simulation and database sequence analysis, and launched the in-depth humanization service of antibodies. Methods: The in-depth antibody humanization service integrates the technologies of non-human sequence 3D structure simulation and analysis, eukaryotic expression and purification identification, affinity and functional assay, etc. On the premise of retaining both specificity and affinity of the antibody, the antibodies with more than 95% humanization were eventually obtained. Advantages: Based on computer 3D structure simulation, the antibodies with more than 95% humanized , and back mutations are less than 6. Cases: Since the launch of the service in 2016, SanyouBio has completed more than 200 cases of humanized antibody design, of which more than 40 designed antibodies are in the clinical application stage, and 7 are in the clinical trial stage. Service Contents Service Service Details Client Provides Deliverables Time Mouse antibody humanization 1. humanization design 2. Gene synthesis 3. Vector construction 4. Eukaryotic expression 5. Quality control Mouse antibody sequence, target protein and benchmark, cell line for identification, etc Deliverables: humanized sequence, protein, plasmid and Nanobody test report; Humanization > 95%, at least one antibody affinity is similar or better than the rabbit antibody sequence, parental antibody 4-6 weeks Nanobody humanization Nanobody sequence, target protein and benchmark, cell line for identification, etc Rabbit antibody humanization Rabbit antibody sequence, target protein and benchmark, cell line for identification, etc Service Highlights 1. 3D humanized design and in-depth antibody engineering Based on the 3D structure design and unique humanized antibody database, and guided by a large number of humanized data, the antibodies with more than 95% humanization can be obtained, which can minimize the heterogeneity of antibodies and keep their specificity and affinity unchanged. 2. Unique database and reliable design A unique humanized design database is formed based on the rich project design and validation data. 3. Eukaryotic expression system, natural modification Using CHO or HEK293 transient expression system, the antibodies obtained have relatively complete post-translational modification. 4. Extensive project experience and high success rate SanyouBio expert team has rich experience in antibody humanization. We have completed more than 160 cases of humanized antibody design, of which more than 40 designed antibodies are in the clinical application stage, and 7 are in the clinical trial stage. 5. Fast delivery in 4-6 weeks and with high quality The one-stop delivery from humanization design, gene synthesis, plasmid construction, protein expression, purification to protein identification only takes 4-6 weeks, which is at the top level in the industry. Case Stastics 1. Mouse antibody sequence 3D structure simulation and template analysis 1.1. 3D structure simulation analysis, surface potential analysis, amino acid residue analysis The structure simulation analysis and surface potential analysis of the antibody before and after humanization are shown in the figure below, both of the structure were highly similar. Fig. 1 Structure simulation analysis and surface potential analysis 1.2. Germline analysis and confirmation The mouse antibody sequences were aligned with human germline based on the unique database, and a series of germline templates were selected for mouse sequence site confirmation. Humanized templates are usually based on mouse sequences, and four categories of germline (IGHV1/3 and IGKV1/3) with good druggability were selected as design templates. 1.3. Immunogenicity prediction of mouse antibody sequences Based on the public database, the immunogenicity of the peptide binding to immune cells in mouse antibody sequences was analyzed. 1.4. Prediction of post-translational modification sequence sites The post-translational modification sites were predicted and analyzed with reference to the unique database. Modification Heavy Chain Variable Region (VH) Light Chain Variable Region (VK) Result Low Risk Medium Risk High Risk Low Risk Medium Risk High Risk Glycosylation / / / / / / Low Risk Glycation / K19 / / K49 / Medium Risk Oxidation W47 M3/M92 / / / / Medium Risk Deamidation / N56 / / / N76/N90 High Risk Isomerization D61 / / / / / Low Risk Fragmentation / / / / / / Low Risk Free Cystine / / / / / / No Free Cystine 2. Eukaryotic expression, purification and identification of humanized antibody Humanization of mouse antibodies is usually designed with 3-5 light and heavy chain sequences, and 9-25 humanized candidates were generated via orthogonal combination, about 10 candidates were selected for eukaryotic expression verification based on the risk assessment eventually. CHO and HEK293 cells were used as host cell and expressed in 10 mL volume for 5–7 days, The samples (> mg) were obtained by one-step affinity chromatography purification for downstream identification analysis. 3. Quality identification and analysis of humanized antibody 3.1. Analysis of physicochemical properties before and after humanization (SDS-PAGE/SEC/DSF) The physicochemical properties of the humanized antibody, such as SDS-PAGE, SEC and DSF, are consistent with the parent. Fig. 2 SDS-PAGE Purity of antibody protein > 90% Fig. 3 SEC Main peak of antibody protein > 95% Fig. 4 DSF Subject to actual test results 3.2. Binding activity of humanized antibody (Gator / Biacore) The affinity kinetics are indispensable for the evaluation of antibody affinity and druggability. While increasing the proportion of humanization, SanyouBio can also ensure that the antibody affinity activity is basically the same with the parent one. Fig. 5A Affinity kinetic analysis of parental antibody (Gator) Fig. 5B Affinity kinetic analysis of parental antibody (Biacore) Table 1 Gator detection data Antibody KD (M) ka(1/Ms) kd(1/s) R2 Maternal antibody 3.39E-10 6.97E+04 2.36E-05 0.99 Humanized antibody 4.10E-10 5.43E+04 2.23E-05 0.99 Fig. 6A Affinity kinetic analysis of humanized antibody (Gator) Fig. 6B Affinity kinetic analysis of humanized antibody (Biacore) Table 2 Biacore test data Antibody KD(M) ka(1/Ms) kd(1/s) R2 Maternal antibody 3.98E-09 2.12E+05 8.44E-04 0.99 Humanized antibody 2.47E-09 2.48E+05 6.11E-04 0.99 3.3. The binding activity of humanized antibody (FACS / ELISA) The binding and blocking activity before and after humanization of mouse antibody are shown in Fig. 4 and Fig. 5. The EC50 and the binding curves of humanized antibody (HuAb1-8) are highly similar to the parental antibody, which indicates that the cell binding and blocking activity are highly similar to the parent control before humanization. The affinity activity of humanized nanobody is also equivalent to the parent control in Fig.6 and Fig.7. Fig. 7 Comparison of the affinity of humanizedantibodies to parental antibody Fig. 8 Comparison of the blocking activity of humanized antibodies to parental antibody Fig. 9 Comparison of the affinity of humanizedantibodies to parental antibody Fig. 10 Comparison of the affinity of humanizedantibodies to parental antibody 4. Selection principle of humanized antibody The selection principles of humanized candidate antibodies are arranged according to the priorities: 1)The binding or blocking activity (Affinity kinetics are generally three times higher than the parent 2)Degree of antibody humanization (number of back mutations < 8) 3)Expression titer and physicochemical properties (the expression titer and physicochemical properties were consistent with the parental antibody)

Service Contents

Service

Service Details

Client Provides

Deliverables

Time

Mouse antibody humanization

1. humanization design

2. Gene synthesis

3. Vector construction

4. Eukaryotic expression

5. Quality control

Mouse antibody sequence,

target protein and benchmark, cell line for identification, etc

Deliverables: humanized sequence, protein, plasmid and Nanobody test report; Humanization > 95%, at least one antibody affinity is similar or better than the rabbit antibody sequence, parental antibody

4-6 weeks

Nanobody humanization

Nanobody sequence, target protein and benchmark, cell line for identification, etc

Rabbit antibody humanization

Rabbit antibody sequence, target protein and benchmark, cell line for identification, etc


Service Highlights
  • 1. 3D humanized design and in-depth antibody engineering
    1. Based on the 3D structure design and unique humanized antibody database, and guided by a large number of humanized data, the antibodies with more than 95% humanization can be obtained, which can minimize the heterogeneity of antibodies and keep their specificity and affinity unchanged.
  • 2. Unique database and reliable design
    1. A unique humanized design database is formed based on the rich project design and validation data.
  • 3. Eukaryotic expression system, natural modification
    1. Using CHO or HEK293 transient expression system, the antibodies obtained have relatively complete post-translational modification.
  • 4. Extensive project experience and high success rate
    1. SanyouBio expert team has rich experience in antibody humanization. We have completed more than 160 cases of humanized antibody design, of which more than 40 designed antibodies are in the clinical application stage, and 7 are in the clinical trial stage.
  • 5. Fast delivery in 4-6 weeks and with high quality
    1. The one-stop delivery from humanization design, gene synthesis, plasmid construction, protein expression, purification to protein identification only takes 4-6 weeks, which is at the top level in the industry.

Case Studies
1. Mouse antibody sequence 3D structure simulation and template analysis

1.1. 3D structure simulation analysis, surface potential analysis, amino acid residue analysis

The structure simulation analysis and surface potential analysis of the antibody before and after humanization are shown in the figure below, both of the structure were highly similar.


Fig. 1 Structure simulation analysis and surface potential analysis


1.2. Germline analysis and confirmation

The mouse antibody sequences were aligned with human germline based on the unique database, and a series of germline templates were selected for mouse sequence site confirmation. Humanized templates are usually based on mouse sequences, and four categories of germline (IGHV1/3 and IGKV1/3) with good druggability were selected as design templates.


1.3. Immunogenicity prediction of mouse antibody sequences

Based on the public database, the immunogenicity of the peptide binding to immune cells in mouse antibody sequences was analyzed.


1.4. Prediction of post-translational modification sequence sites

The post-translational modification sites were predicted and analyzed with reference to the unique database.


Modification

Heavy Chain Variable Region (VH)

Light Chain Variable Region (VK)

Result


Low Risk

Medium Risk

High Risk

Low Risk

Medium Risk

High Risk


Glycosylation

/

/

/

/

/

/

Low Risk

Glycation

/

K19

/

/

K49

/

Medium Risk

Oxidation

W47

M3/M92

/

/

/

/

Medium Risk

Deamidation

/

N56

/

/

/

N76/N90

High Risk

Isomerization

D61

/

/

/

/

/

Low Risk

Fragmentation

/

/

/

/

/

/

Low Risk

Free Cystine

/

/

/

/

/

/

No Free Cystine

2. Eukaryotic expression, purification and identification of humanized antibody

Humanization of mouse antibodies is usually designed with 3-5 light and heavy chain sequences, and 9-25 humanized candidates were generated via orthogonal combination, about 10 candidates were selected for eukaryotic expression verification based on the risk assessment eventually. CHO and HEK293 cells were used as host cell and expressed in 10 mL volume for 5–7 days, The samples (> mg) were obtained by one-step affinity chromatography purification for downstream identification analysis.

3. Quality identification and analysis of humanized antibody

3.1. Analysis of physicochemical properties before and after humanization (SDS-PAGE / SEC / DSF)

The physicochemical properties of the humanized antibody, such as SDS-PAGE, SEC and DSF, are consistent with the parent.


Fig. 2 SDS-PAGE

Purity of antibody protein > 90%


Fig. 3 SEC

Main peak of antibody protein > 95%


Fig. 4 DSF

Subject to actual test results


3.2. Binding activity of humanized antibody (Gator / Biacore)

The affinity kinetics are indispensable for the evaluation of antibody affinity and druggability. While increasing the proportion of humanization, SanyouBio can also ensure that the antibody affinity activity is basically the same with the parent one.


Fig. 5A Affinity kinetic analysis of parental antibody (Gator)


Fig. 5B Affinity kinetic analysis of parental antibody (Biacore)


Table 1 Gator detection data

Antibody

KD (M)

ka (1/Ms)

kd (1/s)

R2

Maternal antibody

3.39E-10

6.97E+04

2.36E-05

0.99

Humanized antibody

4.10E-10

5.43E+04

2.23E-05

0.99


Fig. 6A Affinity kinetic analysis of humanized antibody (Gator)


Fig. 6B Affinity kinetic analysis of humanized antibody (Biacore)


Table 2 Biacore test data

Antibody

KD (M)

ka (1/Ms)

kd (1/s)

R2

Maternal antibody

3.98E-09

2.12E+05

8.44E-04

0.99

Humanized antibody

2.47E-09

2.48E+05

6.11E-04

0.99


3.3. The binding activity of humanized antibody (FACS / ELISA)

The binding and blocking activity before and after humanization of mouse antibody are shown in Fig. 4 and Fig. 5. The EC50 and the binding curves of humanized antibody (HuAb1-8) are highly similar to the parental antibody, which indicates that the cell binding and blocking activity are highly similar to the parent control before humanization. The affinity activity of humanized nanobody is also equivalent to the parent control in Fig.6 and Fig.7.


Fig. 7 Comparison of the affinity of humanizedantibodies to parental antibody


Fig. 8 Comparison of the blocking activity of humanized antibodies to parental antibody


Fig. 9 Comparison of the affinity of humanizedantibodies to parental antibody


Fig. 10 Comparison of the affinity of humanizedantibodies to parental antibody

4. Selection principle of humanized antibody


The selection principles of humanized candidate antibodies are arranged according to the priorities: 
1) The binding or blocking activity (Affinity kinetics are generally three times higher than the parent 
2) Degree of antibody humanization (number of back mutations < 8) 
3) Expression titer and physicochemical properties (the expression titer and physicochemical properties were consistent with the parental antibody)