The Use of Light-Scattering Detection with SEC and HPLC for Protein and Antibody Studies, Part I: Background, Theory, and Potential Uses - - Chromatography Online
The Use of Light-Scattering Detection with SEC and HPLC for Protein and Antibody Studies, Part I: Background, Theory, and Potential Uses


LCGC North America
Volume 30, Issue 9, pp. 842-849

Characterizing Drug Conjugates

Another very important and growing area of biotechnology characterization relates to antibody–drug and protein–drug conjugates, which are synthetically derived combinations of an antibody or protein and a small-molecule drug against a specific disease related to that protein target. Using LS-based techniques, it is possible to measure, with a high degree of accuracy, how much drug (or PEGylation) has been incorporated, on the average of all such species present. It cannot, however, indicate the specific points of such attachments. On the other hand, if these derivatives can be adequately resolved before MS detection, MS can indicate exactly where a drug or PEG molecule is located on a (specific) protein sequence; however, MS cannot accurately measure how much of the drug has been incorporated into all the protein molecules unless each such species can be resolved one from the next. This is yet another area where the capabilities of light scattering are superior to those of MS.

Studying Aggregation

Perhaps the most useful information for the biotech industry is that which indicates the level of aggregation present in any protein or antibody sample, as well as the specific nature of each aggregate (dimer, trimer, and so on), and the R h or R g of each aggregate. Another piece of information of value will be a demonstration that the aggregates being measured by SEC with LS detection were indeed present in the original sample, and that their amounts (relative percent peak areas) have not changed through the chromatographic and detection steps.

Light scattering by itself does not change the nature of aggregation in any given sample, but that may not always be true for SEC and HPLC or other modes of separations, or when using MS detection methods. At times, SEC and MS have both been shown to change the nature of aggregates detected after initial injection into a separation-MS scheme (20–22). Furthermore, the level of aggregates could be made to increase or decrease depending on the conditions used in both the separation (SEC) and MS steps.

This, of course, is not a desirable occurrence. If an analytical method changes the original sample components, then it becomes useless in determining what was originally in the sample before analysis. Analysts must demonstrate using accepted methods that their analytical methods have not in any way altered the original samples through the analytical scheme. If this cannot be done, then one should assume the worst: that there are indefinable alterations of the sample components during that analytical method. In essence, that makes all data and conclusions questionable at best and, in reality, worthless.

Even gel permeation chromatography (GPC) and SEC are known to change sample composition, especially in the case of noncovalent aggregation. (In the case of covalent aggregates, other than for disulfide rearrangements, there are usually no changes in the sample composition by HPLC or ultrahigh-pressure liquid chromatography [UHPLC] methods.) In the case of a complex mixture of synthetic, organic, or natural polymers, changes in elution times because of the particular GPC or SEC conditions being used may suggest differing molecular weights as a function of concentrations injected, which is artifactual. This concentration dependence will mainly occur when using a reference calibration method but should not occur when using an absolute technique such as light scattering. Of course, if some of the injected biopolymer mixture remains on the SEC column, then light scattering will provide the true molecular weight for what is being eluted but not necessarily what was in the sample injected. Knowing when this is happening is, of course, extremely important in characterizing the original sample.

In part II of this column series, we provide further discussion of some of the possible pitfalls in attempting to characterize noncovalent protein aggregates using today's MS methods. Such approaches can be fraught with danger, unless the analyst has very clearly and definitively demonstrated that his or her methods do not and will not alter the sample components or their relative amounts.

Today, most applications of light scattering in the biopharmaceutical industry are done using MALS–RI–UV combinations, or some other type of instrumentation arrangement (such as RALS–LALS).


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