Spontaneous reactions of the hydrazone

Spontaneous reactions of the hydrazone

Offering bioconjugation expertise with consistent finesse

When it comes to linking two biomolecules together, the SoluLINK® technology offers a user-friendly solution with reliable and reproducible bioconjugation, which is a great option for what may initially seem to be a daunting task. However, there is always a team of scientists behind the scenes ready to guide you with the kits and reagents, or for a consult when you need more assistance to develop conjugation strategies for routine, novel, and custom applications. Jim Williams serves as the Associate Director of Bioconjugation Technology, where he oversees bioconjugation product manufacturing and is highly involved in the design and manufacturing of custom and OEM conjugates.

Jim brings his biochemistry and molecular biology expertise with him. This knowledge base provides a solid foundation when developing bioconjugation kits and reagents and extending their use into novel applications. With hands-on conjugation experience dating back to the early 2000s, he has performed thousands of conjugations to almost every class of biomolecule and surface available.

Jim Williams: Associate Director Bioconjugation at Vector Laboratories

This has helped him gain an invaluable understanding of the structure, function, and mechanisms of different classes of biomolecules, and he translates that knowledge into bioconjugation kit or reagent development and custom bioconjugate design strategy.

"... I came on board to teach the newer associates about bioconjugation and the considerations for custom conjugate development. Now, as Associate Director of Bioconjugation Technology, I oversee the manufacturing of bioconjugation kits and stand-alone linking reagents and am highly involved in the design and manufacturing of custom and OEM conjugates for global biotechnology companies.”

Researchers often face challenges that require innovative approaches. As Jim explains, bioconjugation can be a portal to applications that otherwise might be limiting. A bioconjugate can be used for applications in which neither individual component could be used effectively on its own. For example, an antibody may be optimized to bind its epitope, but if it is not conjugated to something — such as an enzyme, fluorophore, or oligonucleotide — there would be no way to tell if the antibody had bound or not, much less quantify the amount of analyte present.

"There are many reasons why a researcher would need a bioconjugate. In many instances, as they say, the “whole is greater than the sum of its parts.”

SoluLINK Protein Oligonucleotide Conjugation Kit

But for some researchers, bioconjugation chemistry can be daunting. According to Jim, “Bioconjugation isn’t the “black box” it used to be.” Bioconjugation kits featuring SoluLINK technology are designed for researchers with limited or no bioconjugation experience. These kits include everything needed to successfully conjugate biomolecules according to an optimized, easy-to-use protocol. Since separating the resulting conjugate from unreacted starting materials can be a critical assay step to increase signal-to-noise ratios and provide optimal results, most of the kits also include the materials needed for purification. All of this differentiates kits containing SoluLINK technology from other bioconjugation kits on the market today.

"Other kits leave the researcher wondering whether their reaction worked or not, as there is absolutely no information about the chemistry employed nor is there any way to measure the molar substitution ratio of the linker or the degree of conjugation.”

“When starting a conjugation reaction with only 100 μg or less of material, preserving precious starting material at every step becomes an important consideration to maximize yield”, explains Jim. The efficiency of SoluLINK technology and the two linkers at the heart of every kit and custom conjugate — HyNic and 4FB, which spontaneously and rapidly form bis-aryl hydrazone — are critical to delivering high-yield conjugates. He attributes the discovery by Dirksen and coworkers, which demonstrated that hydrazone reaction kinetics could be accelerated up to 100-fold with catalytic amounts of aniline, to the extremely fast reaction kinetics of conjugate formation. This enabled the production of kits where not only the length of the conjugation step could be reduced from a day or more to merely a couple hours, but where there could also be a complete conversion from starting material to conjugate.

Another of the built-in advantages of the bis-aryl hydrazone is that it is chromophoric – absorbing maximally at 354 nm with a large extinction coefficient (29,000 Lmol-1cm-1). These characteristics allow real-time quantification using a spectrophotometer to visualize both the degree of conjugation and the number of linkers incorporated on each biomolecule prior to conjugation.

HyNic & 4FB Structure

This feature, unique to SoluLINK technology, allows for unprecedented control over the conjugation reaction, with the ability to tailor the linker molar substitution ratio before the conjugation begins and use real-time monitoring to stop or “quench” the reaction once a pre-determined absorbance is reached. This type of control enables the ability to reproduce the same degree of conjugation every time.

“Since every custom conjugate is different, our first step is to set up a technical call to discuss the project. We then recommend an optimal approach to generate the conjugate with the greatest performance for a particular application. For instance, an antibody-HRP conjugate intended for use in ELISA would be prepared differently from a conjugate intended for IHC. We view each conjugation project as a collaboration... I think of the interactions as more of a concierge service rather than just a simple order that needs to be fulfilled.”

When Vector Laboratories receives a request from a customer for help with scale up of a reaction beyond the capacity of existing kits or to meet any other custom need, the Bioconjugation Team readies for action. For example, when a customer needs up to grams of conjugate, which is beyond the scale of the traditional kits, experienced in-house bioconjugation experts work to help develop a plan to meet the request. And, when there isn’t a kit available to produce a specific conjugate, Vector Laboratories can provide a convenient and reliable way to meet the customer’s need with the conjugate produced on site and a certificate of analysis reporting on the analytical techniques (FPLC and HPLC, LC-MS, PAGE, etc.) to characterize that product.

Jim isn’t surprised by how far bioconjugate technology has come or by the future potential yet to be realized by SoluLINK technology. Antibody-oligo conjugates in use today are able to accurately measure analytes in the low pico- to femtomolar range using PCR with excellent specificity. Additionally, they have enabled multiomics assays that simultaneously measure biomarker abundance in single cells and tissues, coupled with mRNA and genomic DNA sequencing, delivering an unprecedented amount of data on diseased and normal cell function. Jim surmises that antibody-oligo conjugation technology is already changing the therapeutic and diagnostic landscape; he believes that the utility of bioconjugates is limited only by imagination.

September 8, 2021
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