Bioconjugation is a chemical reaction that connects two or more organic molecules together to create a new molecule. In this article, we use the term to describe those chemical processes that alter the properties of a biomolecule through conjugation, immobilisation, covalent modification, or labelling.
What Are Some of the Most Important Safety Concerns When Performing a Bioconjugation Reaction?
There are several issues to take into consideration during a bioconjugation reaction, and at places like Fleet Bioprocessing, they are very important to take into account before doing anything.
1. Are the Functional Groups in a Biopolymer Accessible?
Biopolymers are larger molecules featuring secondary, tertiary or quaternary structures. This means that not all functional groups in a polymer are accessible to the modifying agents. In order to expose all of the functional groups in a polymer, operators resort to manipulations such as adding salt, detergent, or even modifying the pH. Nevertheless, you need to ensure that these alterations don’t lead to a denaturation of your polymer.
2. What is the Molar Ratio of the Reactants?
In conventional chemical reactions, the stoichiometry of the reaction results from the molar ratio of the reactants. Consequently, whenever two compounds need to be covalently coupled, the reaction would require approximately equimolar amounts of the initial reagents. Nevertheless, when it comes to biopolymer conjugations, the molar ratio of the reactants depends on your desired degree of the conjugation, but also on whether or not the starting materials are available in the required amounts. For instance, if the intention is to alter a biopolymer by conjugating it with a small molecule, you’ll have to use a much higher amount of the small molecule in order to ensure the completion of the chemical reaction that will occur.
3. What Are the Concentrations of Your Reactants?
Most biomolecules naturally occur at very low concentration. Also, commercial biomolecules may only be available in low concentrations. This is why effective chemical reactions may require very high reaction rate constants by comparison with traditional organic synthesis as you know it. The best way to enhance the reaction rate in such situations is to concentrate your biomolecules prior to initiating a bioconjugation reaction.
4. What is the Characterisation of Your Bioconjugation Reaction?
Traditional organic reactions involve the analysis of a small molecule. On the contrary, when it comes to biopolymer conjugation reactions, all conventional methods such as IR, TLC, HPLC, and NMR may fail. Oligonucleotides, peptides and other such small biopolymers may be suitable for monitoring by HPLC and LC-MS. However, larger biopolymers such as proteins require monitoring by size exclusion column chromatography or gel electrophoresis.
5. What is the Complexity of the Reactions?
Traditional chemical reactions have a high yield, high reproducibility, and simple products. On the contrary, a biopolymer conjugation usually has a low yield, and isn’t always reproducible. Also, it involves extremely complex products. This makes all things much more complex, and therefore calls for an exhaustive analysis of all elements. A particular reaction condition that suits a specific biopolymer may fail to work for another similar biomolecule.
6. What is the Characterisation of Bioconjugates?
The most common method to measure the purity of bioconjugates is gel electrophoresis. In some situations, when the biopolymers are relatively small, the molecular weight can be measured by mass spectroscopy. However, this method requires a crystal structure or the use of a single functional group for conjugation. Unless these conditions are met, the results may not be too accurate.