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Antibody Optimization
Discovering finely-tuned antibody through Affinity Maturation and Antibody Humanization
Overview
Antibody optimization is an important and necessary process to develop antibodies for therapeutic application. The preliminary hits screened from the antibody library are generally hard to have multiple required properties at once such as affinity and stability. To become product candidates, the early antibody candidates need to be engineered to enhance their good properties and reduce hurdles to further develop.
Y-Biologics provides a one-stop antibody optimization process including Affinity Maturation and Antibody Humanization. Our dedicated science team with expertise offers specific suggestions and accurate guidelines considering the developmental purpose of clients. To increase the chance of success, we utilize extensive antibody databases accumulated for a decade and our proprietary fully human antibody library, Ymax® -ABL(See more) as a back born structure to construct antibody libraries for the needs of the clients.
Affinity Maturation

Affinity Maturation is the process to increase antibody affinity, avidity and stability for an antigen through multiple rounds
of somatic hypermutation and selection in the germinal center in vivo. In the laboratory, scientists apply Affinity Maturation
to lead antibodies generated from the antibody library by introducing mutations and selections with phage display technology in vitro.

Y-Biologics offers tailored Affinity Maturation service using diverse methods such as Light chain shuffling,
Structure-based mutation and CDR hot spot mutation. We apply these techniques alone or together after consulting
with a client to maximize the chance of success.

Parent antibody
Low affinity
Affinity Maturation
Selection
In 23 weeks
Optimized antibody

High Affinity

Affinity Maturation Techniques
Light chain shuffling

This approach maintains the heavy chain of a parental antibody.
We insert the amplified heavy chain to a light chain pool which has 106~107 diversity.
As we construct the library for Light chain shuffling and perform biopanning followed by a selection of positive clones against the target.

We analyze and predict CDR residues in the heavy chain which are presumed to be important binding sites with the targets.

We introduce random mutations by substituting those residues with our proprietary antibody library to increase the affinity and construct the library for biopanning with phage display technology.

Structure-based mutation

CDR hotspot mutation 

Somatic hypermutation occurs in CDR Hotspot which is located in the variable region of the heavy chain in vivo.
We apply random mutations in those hotspot regions by utilizing our proprietary antibody library as a backbone and generate the library followed by antibody screening against the target
Points of Differentiation
Multiple Strategies
Increase the probability to discover your candidate using multiple strategies
Highly experienced
Extensive antibody database and assigned a PhD manager and skilled scientists

Great Diversity of Light Chain Pool

We have light chain pool with exceptional diversity 1×107

Successful example

We have successfully optimized various lead antibodies screened from our fully human antibody library, Ymax-ABL®(See more), to become a candidate antibody which is listed up in our pipeline as YBL-006 through Affinity Maturation. The pipeline has been entered the clinical phaseⅠin Australia and South Korea in the current state.
Parental
Affinity Maturation-1
Affinity Maturation-2
Clone name

KD (M)

Koff (1/Ms)

Kdis (1/s)
Fold increase
SA0550 (parental)
3.3ⅹ10-9
5.2ⅹ104
1.7ⅹ10-4
SA1616 (Affinity Maturation-1)
2.2ⅹ10-10
3.1ⅹ105
6.8ⅹ10-5
15
SA1611 (Affinity Maturation-2)
9.2ⅹ10-11
2.3ⅹ105
2.1ⅹ10-5
36
Timeline of Affinity Maturation
4 weeks
Library construction for Affinity Maturation
4 weeks
Bio panning
4 weeks
Screening
2 weeks
Sequencing
5 weeks

IgG Reformatting & Production

4 weeks
Binding Kinetics Analysis
2 weeks
Final Report
Customer preparation: Protein(s) with different tags
Deliverables: Final report, Antibody variable region sequences, purified IgG antibody(If a client requested)
  • There may be changes depending on the conditions of the experiment
  • Details can be coordinated at each stage, so please contact us for more information.
  • There will be an additional cost for the overseas shipping fee.
Antibody Humanization

Antibody Humanization is a process of replacing non-human antibody frameworks and CDRs with human ones.
Antibodies derived from non-human species cause several immune responses, so they should be designed to be safe in the human body for the development of therapeutic purposes. The aim of this process is for eliminating xenogeneic residues but should not be related to the target recognition.

Y-Biologics has developed the Frame-Region Shuffling method which is a proprietary technology of grafting complementary determining regions (CDRs) of a mouse with a fully human antibody library, Ymax®-ABL(See more). We construct every single library specialized in Antibody Humanization by the request of clients. The chance to discover specific binders through biopanning may be higher than in-sillico technology because it simulates within a limited range to predict the result value.
  • Mouse
  • Human
Mouse
Humanized
Human
Frame-Region Shuffling method

CDRs are directly related to binding with antigens and framework regions are involved with the structural features. As specifically describe, the binding ability against antigens is determined by SDR (Specificity Determining Residue) in CDR. SDR is usually located in CDR2 & CDR3 of Vand CDR1 & CDR3 of VL. Therefore, when performing CDR grafting as Antibody Humanization, most of companies construct a humanized antibody by grafting mouse CDRs of VH and VL to the human antibody which has similar homology with the mouse antibody.

On the other hand, Y-Biologics performs Antibody Humanization by grafting mouse CDRs to the proprietary fully human antibody library, Ymax®-ABL, and construct libraries for generating humanized antibodies with optimized combination between mouse CDRs and human framework regions.

· Option 1: We substitute all framework regions with the fully human antibody library except for mouse CDRs and then generate humanized antibody by selecting optimized combination. 
· Option 2: We further substitute CDR1 in VH and all framework regions with the fully human library.

After construction of the Ymax VL pool library on the mVH, VL which is the optimal partner is selected firstly. Then, the remaining FRs except for the CDRs of mVH are replaced with the Ymax-ABL library.  The selected VL and library are prepared to generate the antibody which is humanized both both FRs and VL regions excluding only CDRs of VH.  


We perform 3~4 rounds of biopanning against the target after construction of humanized antibody library.
Specific binders are enriched during the biopanning process and this strategy allows us to find humanized antibodies with better physicochemical properties compared with a parental antibody given by the client.

Timeline of Antibody Humanization
4 weeks
Library construction for Affinity Maturation
4 weeks
Bio panning
4 weeks
Screening
2 weeks
Sequencing
5 weeks

IgG Reformatting & Production

4 weeks
Binding Kinetics Analysis
2 weeks
Final Report
Customer preparation: Antigen(s) with different tags
Deliverables: Final report, Antibody variable region sequences, purified IgG antibody(If a client requested)
  • There may be changes depending on the conditions of the experiment.
  • Details can be coordinated at each stage, so please contact us for more information.
  • There will be additional costs for the overseas shipping fees.
Points of Differentiation
Proprietary technologies
Utilizing our fully human
antibody library as a backbone
One-Stop Solutions
Our experts offer foremost
suggestions for your project
Enhanced binding affinity
More chances to find Antibodies improved affinity through biopanning

Successful example

Y-Biologics has efficiently humanized a candidate antibody from our immune-focused library and introduce it to be part of a bi-specific antibody. The humanized bispecific antibody is now in the cell line development stage and licensed out to one of the big pharma companies in South Korea. The productivity of the antibody is quite high and stable even in the bispecific format and which validates the quality and stability of our Antibody Humanization technique.

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