Casey E. Vantucci, Carter K. Asef, Suraj Dhungana, and Mason Chilmonczyk
Andson Biotech, Atlanta, GA
Introduction
Mass spectrometry (MS) is essential for comprehensive characterization of protein therapeutics during biopharmaceutical development. Conventional workflows analyze denatured proteins; however, denaturation can limit insights into biologically relevant structures. Native MS offers significant advantages for assessing higher-order structures, conformational integrity, homogeneity, and multimeric states, yet its adoption for high-throughput applications remain constrained by labor and time-intensive sample preparation and variability of in-house pulled nano electrospray ionization (nanoESI) emitters.
Native protein conformations are preserved by storing them in solutions containing high concentrations of MS incompatible non-volatile buffers. Buffer exchange into MS compatible volatile buffers such as ammonium acetate is essential prior to mass spectrometric analysis. Traditional approaches such as manual offline spin filters or online size-exclusion chromatography are labor intensive and can easily take an hour or more per sample, creating a bottleneck for large-scale studies.
The DynaChip X1™ platform features a microfluidic chip for rapid, automated online buffer exchange via tangential flow filtration and microdialysis. Designed to retain diverse analytes (from 10 kDa to 5 MDa+), the platform efficiently removes high concentrations of salts, metabolites, detergents, and other interferents. With online integrated nanoESI, including the capability to utilize commercially available nano emitters, the DynaChip X1TM reduces native MS workflow time by up to 5x and enables high-resolution characterization of complex biomolecules—including large protein assemblies, mAb glycoforms, protein-ligand complexes, antibody-drug conjugates, mRNA, AAVs, and membrane proteins. This application note explores the cleaning efficiency of the DynaChip X1TM as a nanoESI integrated rapid online buffer exchange system for native MS workflows.
Experimental Methods
NIST Monoclonal Antibody Reference Material 8671 (NIST mAb) was prepared at a concentration of 0.1 mg/mL in different buffer systems and buffer concentrations to assess the online cleaning efficiency of the DynaChip X1TM and offline sample preparation.
MS Instruments
All MS analysis was performed on a Xevo G2 QTof mass spectrometer with a NanoLockSpray ion source (Waters Corporation) or Q Exactive UHMR Orbitrap mass spectrometer with a Nanospray Flex source (ThermoFisher Scientific).
Offline Sample Preparation and Analysis
Buffer exchange was carried out using Amicon 0.5 mL 30kDa MWCO spin filters (Sigma Aldrich, Cat. No. UFC503008) and a 2-wash manufacturer’s protocol. Offline nanoESI measurements were performed using a direct infusion approach assisted by a Chemyx Fusion 200X High-Precision Syringe Pump flowing at 500 nL/min and using a fused silica emitter tip (360 µm OD × 30 µm ID, 15 µm orifice, CoAnn Technologies).
Online Sample Preparation and Analysis
For online buffer exchange, samples were manually loaded into the DynaChip X1TM sample loop (1.2 µL), injected at a flow rate of 500 nL/min (Chemyx Fusion Syringe Pump) using a carrier solvent of 200 mM ammonium acetate, and buffer exchanged into 200 mM ammonium acetate. Online nanoESI measurements were performed using a fused silica emitter tip (360 µm OD × 30 µm ID, 15 µm orifice, CoAnn Technologies).
Results & Discussion
The DynaChip X1TM platform facilitates rapid online buffer exchange, desalting, and removal of interferents as shown in Figure 1.
Figure 1: Schematics of the rapid online buffer exchange by the DynaChip X1TM and integration with nanoESI.
After online buffer exchange, the clean sample was directly introduced to a MS via a coupled nanoESI emitter for analysis within 2 minutes. The integration of the DynaChip X1TM online buffer exchange platform with a MS system provided analytical performance superior to multi-step conventional offline buffer exchange workflows using NIST mAb as a model protein. Native MS analysis of NIST mAb using the DynaChip X1TM -MS integrated system efficiently cleaned 1X PBS buffer and displayed native charge state distribution with fully resolved glycoforms (Figure 2).
Figure 2: Native intact mass analysis of NIST mAb from PBS using DynaChip X1TM online buffer exchange (A,B) or offline buffer exchange (C,D). Total ion chromatogram (A, C) and glycoform resolved MS spectra (B, D) were obtained using each approach.
The glycoform resolved MS spectra for the online buffer exchanged sample using the DynaChip X1TM (Figure 2B) was cleaner than the 2X offline buffer exchanged NIST mAb sample (Figure 2D). Highly resolved glycoforms are indicative of salt adduct free NIST mAb and demonstrated that the DynaChip X1TM provided a clean sample to the MS by effectively removing all of the buffer components. This spectral clarity of the resolved glycoforms was used for the remainder of the study to assess DynaChip X1TM ’s ability to exchange out different buffers and salts effectively. A 5x improved efficiency was observed by streamlining sample preparation and introduction into a single step using the DynaChip X1TM . This standard configuration provided a spray time of more than 5 min, enough to perform multiple native MS experiments.
Table 1: Summary of Buffer Component Cleanup. Note: Components are evaluated individually or as a mixture, and the highest concentrations listed are those successfully removed by the DynaChipTM to date. Higher concentrations have not yet been tested in all cases.
NIST mAb samples prepared in different salts, buffers, and detergents at various concentrations were analyzed using the DynaChip X1TM online buffer exchange system integrated with a MS. Table 1 summarizes different buffer components evaluated either individually or as a mixture along with the highest concentration of buffer component tested to date and effectively removed by the DynaChip X1TM . The data in Table 1 support that the DynaChip X1TM is very efficient at rapidly exchanging buffers and removing salts and clearing out select detergents from protein samples prior to MS analysis. A critical advantage of the online approach is the delivery of sustained nanoESI spray via the DynaChip X1TM platform, which significantly streamlines the native MS workflow by eliminating manual sample preparation and introduction into the MS. This results in minimal dilution and sample loss. By minimizing manual handling and reducing sample loss, the workflow enhances throughput, reproducibility, and data fidelity, making it an attractive next-generation online sample preparation system for native MS workflows.
Conclusions
The DynaChip X1TM online buffer exchange platform rapidly and efficiently removed interferents, salts, and detergents from different buffer systems and allowed for a streamlined native MS workflow by direct nanoESI integration.
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LIT-AB-BE-26-001 _v1
