Size-based protein separation by CE(SDS) is indispensable for process and quality control in the biopharmaceutical industry and is commonly applied for purity assessment on behalf of drug approval. To overcome the challenge of uncertain peak identification of CE(SDS)-separated fragments and impurities, here, the first online hyphenation with mass spectrometry is presented. This hyphenation is achieved by introducing a second electrophoretic, MS-compatible CZE dimension after the first generic CE(SDS) separation. Both dimensions are hyphenated via a 4-port nanoliter valve that is commercially available from VICI AG International. As soon as the peak of interest of the CE(SDS) separation reaches the sample loop, the separation is stopped. Due to the very stable SDS-protein complex that can not be separated electrophoretically in the CZE dimension alone, a decomplexation with a solvent and a cationic surfactant is required. Therefore, simultaneously to the separation in the 1D, methanol as presample and CTAB (solved in methanol : water (1:1)) as postsample zone are injected and positioned via a C4D detector in the second CZE dimension. The peak of interest is then transferred via a heart-cut approach between the decomplexation zones in the 2D. With this set-up, it was possible to demonstrate the proof of principle. Universal application of the mass spectrometric CE(SDS) protein characterization is presented by soybean protein analysis. By analyzing several mAb fragments, the suitability and power of the 2D system for pertinent biopharmaceutical questions are proven. The intact mass determination facilitate identification of these fragments directly from generic CE(SDS) separation for the first time. In addition, the analysis allows a deeper insight, revealing migration order shifts and detection of several fragments, migrating simultaneously in the CE(SDS) dimension. Detection of these proteoforms resulting from only one CE(SDS) peak demonstrates the necessity of a more detailed investigation that can only be achieved by mass spectrometry hyphenation.
One major drawback of this 4-port nanoliter valve approach is the system and measurement instability due to frequently occurring current leakage. This current leakage is mainly related to the utilized 4-port nanoliter valves material and design, with distances of less than 1 mm between the separation dimensions. Additionally, the exact positioning of the CE(SDS) peak from the 1D in between the decomplexation zones in the 2D is crucial for successful decomplexation and directly related to MS signal intensity. For these main reasons, further development of the nanoliter valve and method adaptation was the primary aim of the second part of this work to improve system and measurement stability. Therefore, an 8-port nanoliter valve was developed and designed.
To allow immediate and unambiguous identification and characterization of CE(SDS)- separated peaks, the optimized system was hyphenated with top-down mass spectrometry. After successful hyphenation of the 2D system with the Orbitrap MS via nanoflow sheath liquid interface, method compatibility of the developed top-down MS method with the decomplexation strategy was tested. It was possible to prove that concentration down to 0.03 mg/mL of NIST mAb LC can be analysed by top-down MS analysis. Furthermore, the presence of intra disulfide bonds was verified by intact mass determination and MS2 fragmentation. In the last part, the identification and characterization of stress-induced fragments of two different mAbs were performed. Both mAb fragments were identified as Fc fragments of the HC, and the cleavage mechanism could be suggested. This identification was only feasible by MS2 top-down measurements and not possible by intact mass analysis alone, representing the value of the third part of this work.
The here presented work demonstrates the first online mass spectrometric characterization of generic CE(SDS)-separated proteins. It is shown that intact mass determination strongly supports the identification of these proteins and mAb fragments. The observation of unexpected migration order shift and several masses of proteoforms by analyzing only one CE(SDS) peak confirm the ambiguity of optical detection alone. Top-down experiments verify the strength of direct identification of mAb fragments and PTMs like the presence of disulfide bonds, which can not be distinguished by intact mass determination alone. This direct and unambiguous identification of CE(SDS)-separated proteins provides a detailed insight. It facilitates the understanding of protein stability and fragmentation, thereby supporting the production, safety and efficacy assurance of biopharmaceuticals
