A chemical process in which two or more than two biomolecules are linked to together for the creation of new molecules makes for the strict definition of bioconjugation. Here, we use the term “bioconjugation” for describing any chemical process that is involved in changing the properties of a biomolecule through immobilisation, conjugation, labelling, or modification. Looking at assay development companies will give you further insight.
Key concerns for the performance of a bioconjugation reaction
While performing a bioconjugation reaction you need to keep several things in mind.
The accessibility that a biopolymer’s functional groups have: Large molecules that have structures that can be secondary, tertiary or quaternary are called polymers. In a biopolymer, at times, the functional groups cannot be accessed by modification agents. To expose these functional groups, manipulations are carried out by changing the pH, or by adding salt or detergents. Even so, it is important to ensure that there is no denaturing of the biopolymer under the changed conditions.
Reactants Molar Ratio
In conventional chemical reactions, the stoichiometry of the reaction is a typical reflection of the molar ratio of reactants. As a result, in a simple reaction in which two compounds are to be combined covalently, it is necessary to use the starting agents in equimolar amounts.
However, in biopolymer conjugations, this molar ratio of reactants is largely dependent on the starting materials that are available and the degree of conjugation desired. For any reaction for the modification of a biopolymer to conjugate it with a smaller molecule, the starting biopolymer is usually limited. A large excess of the smaller molecule can then be used to drive the reaction to completion.
Biomolecules exist naturally in most cases as very low concentrations and the availability of commercial biomolecules is restricted to lower concentrations. As a result, you require higher reaction rate constants, for any effective reaction, when you compare it with an organic synthesis that is traditionally carried out. To enhance reaction rates, before attempting bioconjugation reactions, it can help if biomolecules are concentrated.
Bioconjugation reaction characterisation
Standard characterisation methods like HPLC, TLC, IR, and MMR may not work in biopolymer conjugation reactions, as they will in the analysis of small molecules subjected to conventional organic reactions. It is possible to monitor relatively smaller biopolymers, like oligonucleotides or peptides, through LC-MS and HPLC. Larger polymers like proteins will require monitoring through size exclusion column chromatography or electrophoresis.
In conventional chemical reactions, yields are high, the product is simple, and the reaction can be reproduced, whereas, by contrast, yield in biopolymer conjugations is low, products complex, and contain poly conjugated products besides other products and isoforms. A reaction condition that works for one biopolymer may not work for another biopolymer, even if it is similar.
For assessing the purity of bioconjugates, typically, use is made of gel electrophoresis. For a biopolymer that is not very massive, use can be made of Mass Spectroscopy for determining its molecular weight.
When a crystal structure is unavailable, or where single unique functional groups are not used for conjugation, it becomes difficult to know the molecular ratio of reactants and the exact conjugation site. Because of this difficulty and complexity, the effort needed for assessing the purity and composition of a bioconjugate will depend on the rigour and requirements of the downstream research.