Custom Antibody Services
Antibody Affinity Maturation Services
Improve antibody affinity with optimized affinity maturation services. By leveraging our STage Enhanced Maturation (STEMTM) platform, you’ll maximize your chances of identifying the best therapeutic or diagnostic lead candidates for even the toughest targets.
Overview
We offer our affinity maturation services as part of our end-to-end antibody development services. R&D teams can get high-quality antibodies with our flexible, optimized, and validated approach that improves affinity, specificity, functionality, and developability.
Our success rates are high, and it all comes down to our extensive experience, fully optimized processes and innovative technology:
STEMTM (STage-Enhanced Maturation) is our multi-stage antibody affinity maturation platform that mimics the natural immune system to engineer enhanced function. By iterating over multiple stages, we can explore a sequence landscape vastly larger compared to traditional approaches. And by using directed evolution to constrain diversity, instead of artificial parameters designed in silico, we ensure that each antibody possesses the functions and characteristics you need.
After each engineering stage, we characterize the lead candidates and discuss the project outcomes with you before proceeding to the next stage. This allows us to quickly adapt our engineering approach and saves you time and resources by avoiding unnecessary steps or dead ends.
To get started designing your libraries, all we need is the amino acid sequence of your antibody!
| Stage 1 | Stage 2 | Stage 3 | 6 CDR-targeted libraries | L1+L2+L3 and H1+H2+H3 combined in separate libraries | Combined H+L |
|---|---|---|
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| Six separate libraries target each CDR individually. Targeted residues are chosen based on our extensive antibody engineering experience. Selection focuses the libraries to functional CDR sequences. | Two separate libraries are constructed, combining the pre-selected CDR pools from the heavy or light chain libraries. Selection identifies optimized mutations that work across CDRs. | The final library pairs pre-selected heavy and light chains. Only optimized clones that possess the highest affinity and that survive developability screening are selected. |
Benefits of Phage Technology for Affinity Maturation
Phage selection is extremely versatile and can be tailored to improve specificity, affinity, thermostability, and pH sensitivity to get you high-quality antibodies as early as possible. Unique in vitro selections enable on-rate and/or off-rate targeting for kinetic optimization. We also have extensive experience phage panning on native or transfected cells as antigens.
High specificity & affinity
With decades of collective experience, Abwiz Bio has led numerous successful affinity maturation campaigns, producing >10-fold affinity improvement for human, mouse, and rabbit antibodies with low nanomolar starting affinity.
More diversity & high yields
Our approach covers more diversity and yields higher affinity clones that cannot be achieved by traditional methods such as CDR walking. Phage libraries are easily >1E10 in size, compared to 1E8 maximum size of yeast libraries.
Ensured developability
Developability is integrated into all selections. Thermoresistance selection and BVP subtraction are used during phage panning to remove unstable, polyreactive clones possessing poor developability profiles.
The STEMTM protocol has been robustly validated
With STEMTM , you maximize sequence diversity, so you have more opportunities to find top-tier lead candidates. In fact, our 3-stage engineering strategy was used to improve affinity for an antibody lead candidate that recognized a high-profile cancer target. The sequencing analysis of the engineered clones revealed unique CDR sequences at each stage—reflecting diversity and providing strong support of our novel STEMTM approach.
Our approach captures these unique sequences by combining the entire pre-selected CDR pool for additional library selection. This is unlike traditional CDR walking protocols, which are designed to choose the best CDR clone from a single library before proceeding to engineer the next CDR—limiting the pool of potential sequences and keeping rare, but important, clones hidden.
The number of unique CDRs identified during screening is shown below. (# of unique CDRs)/(total # of CDRs) shows the sequence diversity of the selected clones for each CDR at each screening stage.
| CDR | H1 | H2 | L1 | L2 |
|---|---|---|---|---|
| Stage 1 | 24/24 | 23/24 | 24/24 | 23/24 |
| Stage 2 | 49/56 | 52/56 | 47/50 | 36/50 |
| Stage 3 | 74/82 | 44/82 | 76/82 | 64/82 |
The number of unique CDRs identified in previous selection rounds is shown below. This shows the number of clones that possessed a CDR sequence previously identified in Stage 1 (or in Stages 1 and 2).
| CDR | H1 | H2 | L1 | L2 |
|---|---|---|---|---|
| Stage 1 | - | - | - | - |
| Stage 2 | 0 | 1 | 0 | 7 |
| Stage 3 | 0 | 16 | 8 | 18 |
Our affinity maturation services are flexible—adjusting to your needs
We understand time is of the essence and our goal is to efficiently deliver the antibodies you need, so we’ve made our affinity maturation services flexible and data-driven. We work closely with your team at the beginning of every stage to carefully review prior screening data and to develop a solid plan. If you are satisfied with the affinity of the antibodies at the end of stage 1 (single CDR libraries), no need to continue with stage 2 and 3—saving you time and resources.
In one project, our client was satisfied with the affinity improvement after Stage 2 (heavy or light chain only libraries). We opted to forego Stage 3 (combined heavy and light chain libraries), allowing the client to start using the antibodies as early as possible.
Stage 2 clones were tested against parental wild-type Fab and the best Stage 1 engineered clones in off-rate ELISA. The mutations selected from Stage 2 libraries improved affinity substantially.
Success relies on a carefully designed library
Your success is our success, and antibodies can only be as good as the library. With extensive experience in affinity maturation of therapeutic and diagnostic lead candidates, we know what library strategies work best. Our goal is to maximize library diversity while maintaining developability so you get functional, scale up-ready antibodies.
We target each CDR in a separate library to optimize antigen interactions for each individual loop in Stage 1.
Affinity can be improved by targeting the heavy and light chains of the CDRs for mutagenesis. However, the biological constraints of phage display (limits on maximal E. coli transformation efficiency) restrict the total number of possible variants within a given library to ~1E+10 (Much larger library size compared to yeast display, with a maximum of ~1E+08). Randomizing a stretch of six amino acids to all possible variants creates a theoretical diversity of 1.07E+09; thus theoretical diversity can exponentially exceed the practical library diversity. And higher diversity means more options to choose from.
We only choose mutations that will be tolerated by the structure
We determine tolerability based on:
- Germline comparison – which residues are different?
- Comparison to published antibody structures (bioinformatics analysis)
- Which positions show high variability and which are conserved?
- Our prior experience (rabbit, mouse, human mAbs) – which residues tolerate mutation?
We use synthetic oligos to synthesize libraries with precision
Synthetic oligos target CDRs using degenerate codons, which allows accurate and precise control over library composition.
Timeline
| Steps | Time | |
|---|---|---|
| Library Construction | 4-6 weeks | |
|
•Gene/oligo synthesis •Library synthesis •Pilot studies |
2-3 weeks 2-3 weeks 1 week (concurrent) |
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| Panning/Screening | 2-3 weeks | |
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•Phage panning •Fab screening •Sequence analysis |
1 week 3-5 days 2 days |
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| Stage 1 Completion | ~2 months after gene synthesis | |
| Stage 2/3 | Informed by Stage 1 results | 4-6 weeks |
Pricing
FAQs
STEM uses pre-selected, functional CDR libraries to cover more practical diversity which can be missed when using alternative methods such as CDR walking.
The typical steps in CDR walking:
- Mutate a single CDR
- Identify the best clone
- Use this sequence and mutate the next CDR
With CDR walking, only a single CDR sequence is selected per library, keeping paired mutations that work across multiple CDRs from being selected, which ultimately limits affinity improvement.
STEM is a 3 stage approach that allows us to simultaneously mutate CDRs and involves three separate library construction and selection/screening steps. This generates more diverse libraries that capture mAbs with the rarest characteristics—delivering more options for you to choose from.
While we can’t guarantee the outcome of a specific project because every antibody is different, we can guarantee the quality of the mAbs. Each library undergoes QC check by gel electrophoresis and DNA sequencing of numerous colonies, and we validate the mAbs via ELISA, flow cytometry, functional assays, and more upon request.
Because we use phage display, our library sizes are larger than competitors (1E10 compared to 1E8 for yeast) so you have a greater chance of getting the exact characteristics you need. Phage also allows us to select for developability (heat resistance as proxy) so lead candidates can get to the clinic faster.
No. Our affinity maturation services are flexible and tailored to your needs. For our multi-stage STEM process, we can stop at the stage where you are satisfied with the improved antibodies. We work with you at every stage of the process and discuss what our plan is for each subsequent stage to get approval before moving forward.
After each stage of the STEM method, we characterize the Fabs using multiple methods:
- ELISA
- Octet Bio-Layer Interferometry (BLI)
- Flow cytometry
- Functional Assays
- Full sequence analysis
- Surface plasmon resonance (SPR)
Our dual-purpose vector allows soluble Fab expression in 96-well format, so we can screen lead candidates in high-throughput using crude bacterial supernatant before proceeding to the expensive, time-consuming IgG stage.
Yes! Abwiz Bio offers mouse, rabbit, llama, or alpaca antibody humanization as part of its end-to-end antibody development services. Unlike conventional methods, we pair humanization with our affinity maturation technology (STEMTM) to deliver fully optimized mAbs.
Learn more about our Antibody Humanization and Optimization services.
