How to Determine the Isoelectric Point of Antibodies

An important feature of monoclonal antibodies (mAbs) is their isoelectric point (pI), which is essentially the pH value at which the antibody has no net charge, and its value depends on the charged amino acids present in the antibody. If the surrounding environment's pH is lower than the antibody's pI, the molecule carries a net positive charge; whereas when the pH is higher than the pI, the antibody will carry a net negative charge. In evaluating the pharmacokinetic (PK) characteristics of therapeutic antibodies, not only target-mediated drug disposition (TMDD) but also non-target related mechanisms can influence the overall PK behavior, pImay be an important factor in the latter. Moreover, since selecting the optimal candidate drug early in the preclinical stage of product development can greatly reduce development time and costs, it is important to consider risk mitigation strategies and tools during drug discovery and development, including antibody variable region charge and antibody pIanalysis.

Antibody pIdetermination methods are numerous, with common ones including precipitation methods and classical isoelectric focusing electrophoresis. With the advancement of life science technology, isoelectric focusing electrophoresis has also been improved, such as whole-column imaging capillary isoelectric focusing (icIEF) and pressure-mediated capillary electrophoresis (PreMCE). A newly developed simple method based on an improved version of PreMCE can be used to determine the isoelectric point of zwitterions with closely spaced pKa. This new pIdetermination PreMCE method (i) can be easily performed on most commercial capillary electrophoresis instruments; (ii) it can use small, impure samples, (iii) unlike isoelectric focusing methods in natural pH gradients, it does not require a linear pH gradient, and (iv) it eliminates errors due to chromatographic retention on the separation chamber walls, pI. Furthermore, the isoelectric point of proteins can also be determined by combining oscillation methods with computer algorithms, using programs written in Visual Basic to calculate the isoelectric point of proteins and other macromolecules with acidic or basic residues, theoretically achieving the desired accuracy of pIvalues, the computer automatically provides the representation of protein charge with pH values, and the corresponding values can also be commanded to be available in tabular form.

Biotage Parker Biotech provides one-stop technical services based on capillary isoelectric focusing (cIEF) for the determination ofprotein isoelectric point, offering advantages over the traditional slab method such as short time efficiency, high accuracy, and low sample usage. This technology is suitable for a variety of amino acids, peptides, recombinant proteins, enzymes, monoclonal antibodies, etc., and has been widely used in clinical analysis. Free consultation is welcome!

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