Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions

Abstract

In the biopharmaceutical industry, CE-SDS assesses the purity, heterogeneity, and stability of therapeutic proteins. However, for mAb-1 and mAb-2, typical CE-SDS under reducing conditions produced atypical protein peak profiles, which led to biased purity results, thus were not acceptable for biologics manufacturing. This bias was caused by the formation of method-induced higher molecular weight artifacts, the levels of which correlated with protein concentration. Here we show that adding sodium tetradecyl and hexadecyl sulfates to the sample and the sieving gel buffer solutions was required to prevent formation of aggregate artifacts and to maintain detergent:protein uniformity, suggesting their importance during the sample preparation steps of heat denaturation and subsequent cooling as well as during capillary migration. For these proteins, we show that this uniformity was likely due to the ability of these detergents to bind proteins with markedly higher affinities compared to SDS. "CE-SC X S" methods (where CE-SC X S is CGE using detergent composed of a sodium sulfate head group and a hydrocarbon tail, with "C X " representing various tail lengths), were developed with a sodium tetradecyl sulfate sample buffer and a sodium hexadecyl sulfate containing sieving gel buffer that minimized artifacts and provided robust characterization and release results for mAb-1 and mAb-2.

Recent developments in capillary and microchip electroseparations of peptides (2019–mid 2021)

“…Replacement of SDS in SDS‐CGE separation of proteins and their peptide fragments by anionic detergents with longer alkyl chain, particularly by tetradecyl sulfate in the sample and hexadecyl sulfate in the sieving BGE, resulted in suppression of artifact protein aggregation in CGE analysis of mAbs under reducing conditions [101]. Heavy and light chains of mAbs could be separated with better repeatability and higher robustness.…”

Elimination of light chain tailing in reducing capillary electrophoresis with sodium dodecyl sulfate analysis of a monoclonal antibody

“…In view of the previous use of STS and SHS to mitigate the HC or intact mAb peak artifacts in CE-SDS [21][22][23], we tried to use CE-SHS and CE-STS to optimize the LC peak shape of mAb-A, that is, replacing the SDS of the sample and gel buffer with SHS and STS, respectively. Both CE-SHS and CE-STS have significantly optimized peak shapes (Figure 5).…”

“…Molina et al [20] reported HMW artifacts caused by incomplete denaturation of mAbs, and they increased the SDS concentration, denaturation temperature, and incubation time to eliminate the artifacts. Guan et al [21][22][23] also reported HMW artifacts in reducing and nonreducing modes. They attributed this phenomenon to the problem of binding between the ionic surfactant SDS and the antibody molecule.…”

“…Guan et al. [21–23] also reported HMW artifacts in reducing and nonreducing modes. They attributed this phenomenon to the problem of binding between the ionic surfactant SDS and the antibody molecule.…”

“…Finally, the use of the longer alkyl chain anionic surfactants sodium tetradecyl sulfate (STS) and sodium hexadecyl sulfate (SHS) as alternatives to SDS achieved the best optimization effect. SHS and STS have been previously reported for the optimization of HC or intact mAbs [16,22,23], but they have never been applied to improve LC peak performance as reported in this article. Here, we proved that SHS was also suitable for eliminating the tailing peak caused by the limitation of the complexation of SDS and LC in reducing CE-SDS, thereby improving the peak shape and providing a more accurate and stable analysis method.…”