As of now, if we want to successfully develop a therapeutic mAb, we need the antibody sequence of this monoclonal antibody to be humanized as well as the affinity of the antibody to be mature. Since the sequence on the surface of the antibody is prone to mutation, it remains unresolved for the antibody to improve its affinity for multiple antigens. In the clinic, researchers often have the idea that if the affinity of an antibody is increased, it may induce higher biological activity in the body, which will positively reduce the amount of injectable drug administered, as well as the possible associated side effects, ultimately reducing pharmaceutical costs. Industrial therapeutic antibodies avoid the need for further in vitro affinity maturation, thus shortening the timeline for antibody development in Tektronix's bio-labs. mAb's instinctive ability to bind target antigens with high specificity and affinity has propelled the role of mAbs in diagnostic and therapeutic applications.

When we introduce mutations during the antibody affinity maturation process, but the introduced mutations do not result in a significant increase in the affinity of our antibodies. The reason for this may be that the mutation method we use is inappropriate, the region of mutation is not selected appropriately, or the frequency of mutation is too low in the process of antibody affinity mutation. While selecting the highest affinity antibody from a large number of mutant antibodies is a difficult challenge, the entire screening process may be interfered by several factors, such as the sensitivity and specificity of the method used in the screening and the stability of the environment in which we conduct the experiments. Teckbio has optimized its mutation strategy to address these issues by combining multiple mutation methods with each other for antibody mutation. We can also utilize the structural sites recognized by antibodies and antigens for sites with a large number of mutated regions. We also use some high-quality products, such as polymerase for high-frequency mutation. In the screening process, Tektronix conducts large-scale screening by using high-throughput screening techniques, such as flow cytometry and surface display technology, which improves both the screening efficiency and the speed and accuracy of high-affinity antibody screening. By establishing a standardized screening system and experimental process to reduce possible errors in experiments, Tektronix conducts in vitro affinity screening to ensure that recombinant antibodies are affinity mature and contribute to the discovery of antibody drugs for our customers.

When we find that antibody stability is not high during the process of antibody affinity maturation, it may be due to the fact that we need to sacrifice the original stability or specificity of the antibody while improving its affinity. It is also possible that the change in antibody structure caused by certain mutations ultimately leads to poor antibody stability, which has an impact on the overall performance of the antibody. In antibody affinity experiments, if the reproducibility of the experiment is found to be poor, the results of the experiment also appear to be relatively large differences between different batches or different laboratories. When Teck Bio conducts mutations, we consider the affinity and stability of the antibody as a whole, which prevents over-optimization leading to a decrease in antibody performance. At the same time, we validate and measure the antibodies selected through multiple experiments to ensure the stability and high affinity of the antibodies in the subsequent applications of our customers. In order to ensure the reproducibility of the experiments, Teckbio reduces the experimental errors from all aspects by strictly controlling the experimental conditions and experimental operations, etc. We have established a perfect experimental record and file management system, which can ensure the authenticity and verifiability of our experimental data.

Mutation strategies for antibody in vitro affinity maturation technology are divided into three main categories: induced random mutagenesis, strand replacement, and directed mutagenesis. Error-prone PCR is a well-established general-purpose random mutagenesis method that takes advantage of the natural error rate of low-fidelity DNA polymerases (i.e., Taq polymerase). mAb identification workflow follows the traditional panning strategy. Desirable mAb clones are selected for affinity maturation and then subjected to error-prone PCR amplification to introduce mutations at the CDR, thereby generating miniature mutant libraries. By manipulating the DNA amplification conditions, ideal mismatches can be generated due to the nature of the polymerase that lacks 3′-5′ proofreading capability. Genetic modification and diversification can be achieved by strand substitution to obtain combinations of mutations beyond what is naturally available. Strand substitutions provide a “mix-and-match” mechanism whereby a library of one strand (heavy or light) is paired with a partner strand (which remains unchanged) to create a second library. This structural domain reorganization can mimic SHM in vivo, thereby increasing affinity through the general effect of the exchanged variable structural domains. Targeted mutagenesis is an in vitro gene modification method involving defined gene loci or specific regions of DNA sequence to study candidate genes at the sequence, structural and functional levels. In order to perform site-specific mutagenesis, structural data are required, which can be derived from previous random mutagenesis studies in which significant mutations and tunable positions were identified.

Antibody affinity maturation is a very complex physiological process that requires the collaboration of several parties. When the body encounters a pathogen invasion, the B lymphocytes of our immune system immediately begin to work, and after a series of signals, our antibody affinity matures. During the process of B cell maturation, the B cells undergo high frequency mutations in somatic cells, generating a rich population of antibody clones, while the B cells are also activated by external antigens through a process mediated by follicular dendritic cells. Together, these mechanisms significantly enhance the average antigen-binding capacity of the B cell progeny and the antibodies they generate, resulting in a natural optimization of antibody affinity in vivo to meet the needs of the human body. When faced with antigenic stimuli, B cells are able to create structural variants of antibodies with the help of genetic rearrangements. Afterwards, CDRs produce high-frequency mutations that enable the antibody to recognize the antigen more accurately.