Single Domain Antibody (SdAb) Stability Improvement Services

Benefiting from their high stability, V_HHs are being utilized for various applications ranging from therapeutics and diagnostics to biotechnology. With our extensive experience and deep understanding of V_HHs, Creative Biolabs has established a unique platform for V_HH stability improvement, which can assist in your V_HH development with the highest efficiency and best quality.

Stability of V_HH

Successful application as therapeutic and diagnostic tools require not only high affinity and specificity but also excellent biochemical and biophysical properties. V_HHs are gaining a reputation as superior recognition elements as they combine the advantages of specificity and affinity with high stability and solubility. Inherently, V_HHs are extraordinarily resistant under harsh and extreme conditions and remain remarkably stable while maintaining full antigen-binding capacity. Most V_HHs exhibit high stability against elevated temperature, high pressure, or chemical denaturants. These unique biochemical and biophysical properties have encouraged their introduction in various applications, such as in vivo immunomodulation, diagnostic detection, and protein purification. Furthermore, their stability under extreme conditions and proteolytic resistance makes V_HHs well-suited for use in alternative routes of administration (i.e., oral administration).

Fig.1 V_HH stability improvement through the introduction of disulfide bonds.^1,3

V_HH Stability Improvement Available at Creative Biolabs

With the remarkable development of V_HHs over the last few decades, extending their application in extreme environments—such as against temperature-induced denaturation, pH-induced denaturation, protease hydrolysis, and other stresses—has become necessary to achieve a variety of research and investigative goals. To address issues of insufficient stability and broaden the application of V_HHs, Creative Biolabs has developed an exceptional platform for V_HH stability improvement, which will be valuable for our customers' V_HH development projects.

Since V_HHs are composed of only a single domain and are small in size, their stability properties can be improved quickly and straightforwardly without impairing their binding properties, allowing them to function under relatively harsh conditions. Our scientists offer stability improvement through rational design of V_HHs or by carefully investigating appropriate strategies, which may be generic solutions or tailored to specific V_HHs for our customers. Our services cover the improvement of both physical and chemical stability, including but not limited to:

At Creative Biolabs, multiple strategies are available to engineer V_HHs with enhanced stability, including but not limited to:

In general, these strategies can be used to efficiently improve V_HH stability while ensuring minimal effects on antigen-binding specificity and affinity. Through these approaches, stabilized V_HHs have been developed that retain their binding ability under extreme conditions, providing superior reagents for a wide range of biotechnology, detection, diagnostic, and therapeutic applications.

Creative Biolabs is a pioneer and undisputed global leader focused on enhancing V_HH stability. As stability is one of the most important requirements for therapeutic and diagnostic applications, Creative Biolabs is pleased to offer V_HH stability improvement services to our clients worldwide. For more detailed information, please feel free to directly send us an inquiry, our scientists are pleased to collaborate closely to promote your project.

Published Data

An Engineered V_HH with Enhanced Protease Resistance and Improved Thermal Stability

Fig. 2 Comparative Thermal Stability of V_HH Mutants under Acidic and Neutral Conditions.^2,3

Fig. 3 Proteolytic Resistance Analysis of V_HH Mutants through Enzymatic Digestion.^2,3

This article examines the enhancement of protease resistance and thermal stability in single-domain antibodies (V_HHs) through engineering approaches. A series of mutants were successfully constructed by introducing two cysteine residues into the V_HHs. Researchers then compared the thermal stability of the modified V_HHs with wild-type V_HHs under acidic and neutral conditions. The results showed that the mutants exhibited higher thermal stability under acidic conditions. In protease digestion assays, these mutants also demonstrated a notable improvement in protease resistance without any discernible impact on biological function. Further research indicated that these V_HH mutants with cysteine modifications displayed enhanced stability and an increased capacity to neutralize toxins, even in environments simulating the gastrointestinal tract. This provides crucial scientific evidence for the development of new oral immunotherapeutic agents.

FAQ

1. How does adding disulfide bonds increase the stability of V_HHs?

The introduction of disulfide bonds creates covalent connections between cysteine residues, reducing flexibility and the likelihood of the V_HH structure unfolding. This stabilization can considerably improve V_HHs' resilience to thermal and chemical denaturation. These connections contribute to the protein's stability under stressful conditions, such as high temperatures or extreme pH, thereby increasing the V_HH's overall resilience and functional lifespan.

2. Can stabilizing excipients enhance V_HH stability?

To prevent deterioration during storage and transportation, stabilizing excipients such as sugars, polyols, and amino acids can greatly improve V_HH stability. These excipients help to stabilize the protein structure, reduce aggregation, and prevent denaturation. For instance, sucrose and trehalose are frequently utilized to maintain the integrity of therapeutic proteins by stabilizing their folded forms and blocking degradation pathways.

3. How is the stability of a V_HH evaluated experimentally?

Numerous biophysical methods can be used to evaluate the stability of a V_HH, including accelerated stability studies under stress circumstances, circular dichroism (CD) spectroscopy for assessing secondary structure stability, and differential scanning calorimetry (DSC) for thermal stability. Functional experiments, such as binding affinity assessments, can also be used to determine stability by demonstrating whether the V_HH maintains its activity after exposure to denaturing conditions.

4. What are the challenges that exist in increasing V_HH stability?

The challenges of improving V_HH stability include maintaining binding affinity and specificity while introducing stabilizing mutations, avoiding immunogenicity caused by sequence changes, and ensuring that stabilization techniques are scalable and cost-effective for industrial production. Balancing these aspects requires a thorough understanding of protein chemistry and the V_HH's specific application requirements.

5. How does pH affect the stability of V_HHs and how can this be addressed?

pH can affect the stability of V_HHs by altering the ionization states of amino acids, potentially leading to distorted interactions or structural changes. To mitigate the impact of pH, V_HHs can be engineered to be stable over a wider pH range by introducing mutations that stabilize the protein at both acidic and basic pH values. Adjusting the buffer during storage and formulation can also contribute to maintaining a stable pH environment for V_HHs.

Resources