Detecting Disulfide Bond Shuffling in Biologics Using Trypsin Platinum

Biologic therapeutics such as monoclonal antibodies and biosimilars are complex proteins that are susceptible to post-translational modifications (PTMs). These chemical modifications can affect the performance and activity of the biologic, potentially resulting in decreased potency and increased immunogenicity. Such modifications include glycosylation, deamidation, oxidation and disulfide bond shuffling. These PTMs can be signs of protein degradation, manufacturing issues or improper storage. Several of these modifications are well characterized, and methods exist for detecting them during biologic manufacture. However, disulfide shuffling is not particularly well characterized for biologics, and no methods exist to easily detect and quantify disulfide bond shuffling in biologics.

Disulfide bond shuffling occurs when the S-S linkage is not between a Cys and its normal partner
Disulfide bonds are important for protein conformation and function

Normally the cysteines in a protein will pair with a predictable or “normal” partner residue either within a polypeptide chain or between two polypeptide chains when they form disulfide bonds. These normal disulfide bonds are important for final protein conformation and stability. Indeed, disulfide bonds are considered an important quality indicator for biologics.

In a recently published study, Coghlan and colleagues designed a semi-automated method for characterizing disulfide bond shuffling on two IgG1 biologics: rituximab (originator drug Rituxan® and biosimilar Acellbia®) and bevacizumab (originator Avastin® and biosimilar Avegra®).

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Why Wait? Sample Prep/Protein Digestion in as Little as 30 Minutes!

While many proteases are used in bottom-up mass spectrometric (MS) analysis, trypsin (4,5) is the de facto protease of choice for most applications. There are several reasons for this: Trypsin is highly efficient, active, and specific. Tryptic peptides produced after proteolysis are ideally suited, in terms of both size (350–1,600 Daltons) and charge (+2 to +4), for MS analysis. One significant drawback to trypsin digestion is the long sample preparation times, which typically range from 4 hours to overnight for most protocols. Achieving efficient digestion usually requires that protein substrates first be unfolded either with surfactants or denaturants such as urea or guanidine. These chemical additives can have negative effects, including protein modification, inhibition of trypsin or incompatibility with downstream LC-MS/MS. Accordingly, additional steps are typically required to remove these compounds prior to analysis.

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Trypsin/Lys-C Mix: Alternative for standard trypsin protein digestions

Trypsin/Lys-C Mix, Mass Spec Grade, is a mixture of Trypsin Gold, Mass Spectrometry Grade, and rLys-C, Mass Spec Grade. The Trypsin/Lys-C Mix is designed to improve digestion of proteins or protein mixtures in solution.It is a little known fact that trypsin cleaves at lysine residues with lesser efficiency than at arginine residues. Inefficient proteolysis at lysine residues is the major cause of missed (undigested) cleavages in trypsin digests.


Supplementing trypsin with Lys-C enables cleavage at lysines with excepetional efficiency and specificity. Following the conventional trypsin digestion protocol (i.e., overnight incubation at nondenaturing conditions, reduction,alkylation, 25:1 protein:protease ratio [w/w], mix and incubate overnight at 37°C.) Replacing trypsin with Trypsin/Lys-C Mix in this conventional protocol leads to multiple benefits for protein analysis including more accurate mass spectrometry-based protein quantitation and improved protein mass spectrometry analytical reproducibility.