Molecular mass analysis of antibodies by on-line SEC-MS

Lowell J. Brady 0 1 2 John Valliere-Douglass 0 1 2 Theresa Martinez 0 1 2 Alain Balland 0 1 2 0 Address reprint requests to Alain Balland, Ph.D., Department of Process and Analytical Sciences , Amgen, Inc., 1201 Amgen Court West, Seattle, WA , 98119, USA 1 Published online January 4, 2008 Received October 23, 2007 Revised December 17, 2007 Accepted December 17, 2007 2 Department of Process and Analytical Sciences , Amgen, Inc, Seattle, Washington, USA Mass analysis of recombinant protein therapeutics is an important assay for product characterization. Intact mass analysis is used to provide confirmation of proper translation of the DNA sequence and to detect the presence of post-translational modifications such as amino acid processing and glycosylation. We present here a method for the rapid mass analysis of antibodies using a polyhydroxyethyl aspartamide column operated in size-exclusion mode and coupled with ESI-MS. This method allows extremely efficient desalting of proteins under acidic conditions that are optimal for subsequent mass analysis using standard ESI conditions. Furthermore, this technique is significantly faster and more sensitive than rpHPLC methods, typically considered the standard chromatography approach for mass analysis of proteins. This method is flexible and robust, and should prove useful for applications where a combination of speed and sensitivity are required. (J Am Soc Mass Spectrom 2008, 19, 502-509) 2008 American Society for Mass Spectrometry - as a mobile phase additive in rpHPLC is often necessary to obtain good chromatography [1], but may result in decreased ionization [10, 11]. Additionally, reduction of antibodies and analysis of the constituent light and heavy chains often results in tailing of the heavy-chain component and the potential for carry-over problems. These issues can lead to lengthened run times or the inclusion of blank runs between samples to increase confidence in the results. We optimized the use of a commercially available polyhydroxyethyl aspartamide column operated in SEC mode (pHEA-SEC) for the rapid on-line analysis of antibodies. This chromatographic material has been originally developed for the separation of polar compounds by hydrophilic interaction chromatography [12]. By operating this column in 0.1% formic acid under isocratic conditions and introducing organic solvent by post-column addition, we developed a robust and fast separation method that is highly flexible and tolerates numerous sample buffer components. Additionally, this new approach differs from other reported SEC-MS methods, achieving a rapid separation in the absence of salts [13] or organic modifiers [14]. We were able to interface this method with an ESI-TOF mass spectrometer and obtained very high quality mass data with experimental to theoretical mass agreements below 25 ppm for several intact antibodies. Furthermore, since this method is highly tolerant of different sample buffer components, we were able to use the same method, without modification, for the analysis of reduced antibody samples. This method is a highly flexible and broadly applicable chromatography procedure for mass analysis of proteins. Mcharacterization assay used to evaluate the ass analysis of recombinant proteins is a key entire amino acid sequence of the molecule and the presence of post-translational modifications. As part of protein drug development, intact mass analysis supports the characterization package for regulatory filings and may be used to evaluate lot-to-lot consistency on a whole molecule level. Mass analysis of intact or reduced antibodies has been used to evaluate the degree of processing of C-terminal lysine on the heavy chain subunit [1]; evaluate N-terminal heterogeneity such as pyroglutamic acid formation [2, 3]; profile N-linked carbohydrate heterogeneity [3, 4]; and to detect instabilities in the molecule such as oxidation [5], succinimide formation from aspartic acid [6], glycation [7], internal cleavage [8], and thioether formation [9]. Reversed-phase HPLC (rpHPLC) separation followed by electrospray ionization-mass spectrometry (ESI-MS) analysis is the typical method for analyzing antibodies under both non-reduced and reducing conditions. This method is highly resolving and advantageous for the detection of minor product impurities and the resolution of different amino acid sequences or heterogeneous post-translational modifications. However, rpHPLC of antibodies has several distinct disadvantages. Due to the large size and relatively hydrophobic nature of antibodies, high temperatures are typically employed to improve elution and peak shape profiles [1, 4]. However, high temperatures may lead to artifactual degradation of the sample during analysis. Inclusion of TFA The CHO-expressed monoclonal antibodies (MAb) used in this study were manufactured at Amgen, Bothell, WA. Column Conditioning and Operation A 2.1 250 mm polyhydroxyethyl aspartamide (pHEA) 300 column received from the manufacturer (PolyLC, Inc., Columbia, MD) was conditioned by operating for 12 to 18 h at 0.2 mL/min on 0.1% formic acid (vol/vol). After this initial conditioning step, the column was operated in the same solvent at 0.1 mL/min at ambient temperature. The column was stored in the same solvent and samples analyzed after operating the column for 10 to 30 min at 0.1 mL/min. For each sample, the column was operated for 8 min as described in the Results and Discussion section. Post-Column Addition of Organic Solvent Acetonitrile with 2.0% (vol/vol) formic acid was added post-column using a stainless steel tee at 0.1 mL/min using an LC Packings Switchos module (Dionex, Inc., Sunnyvale, CA). Any pump capable of providing stable flows of 0.1 mL/min at backpressures up to 20 to 30 bar (due to constriction at the inlet of the mass spectrometer) is appropriate. Sample Preparation and Column Injection Levels Non-reduced samples were analyzed following dilution to 2 mg/mL with water. For reduced samples, the protein was diluted to 2 mg/mL with 20 mM DTT in 6.0 M guanidine HCl/100 mM Tris, pH 8.0 and incubated for 30 min at 37 C. For all samples, an injection of 2 L (corresponding to 4 g) was made onto the column except as noted in the text. The outlet of the mixing tee used to add organic solvent post-column was connected to the internal divert valve attached to an Agilent TOF (model 6210) mass spectrometer (Agilent, Inc., Santa Clara, CA). Key source parameters were as follows: nitrogen gas flow of 9 L/min; fragmenter voltage of 415 V for non-reduced samples and 250 V for reduced samples; capillary voltage of 5000 V for non-reduced samples and 4000 V for reduced samples; and nebulizer pressure of 60 psi. Other conditions were standard ones used with positive ESI for the Agilent TOF. The TOF was calibrated over the range 0 4000 m/z using Agilent ESI calibration mix before analysis with an overall mass error for all points below 3 ppm. Separation by rpHPLC was perf (...truncated)