Rapid Identification and Characterization of Biologics using CESI 8000-TripleTOF MS Platform

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1 Rapid Identification and Characterization of Biologics using CESI 8000-TripleTOF MS Platform High Throughput Qualitative Analysis of Biopharmaceuticals Using CESI-MS Technology Rajeswari Lakshmanan Sciex Separations, Brea, CA Abstract This article describes the analyses of recombinant monoclonal antibodies using Capillary Electrophoresis Electrospray Ionization (CESI) 8000 connected on-line with a TripleTOF mass spectrometer (MS). CESI combines the intrinsic capabilities of capillary electrophoresis (CE) and electrospray ionization (ESI) in a single device, resulting in a robust and highly efficient separation technology with stable spray at ultralow nanolitre flow rates. We have presented a CESI-MS workflow by integrating the CESI separation to TripleTOF, a highly sensitive and accurate mass measurement system for qualitative analysis of biopharmaceuticals. To explore the capabilities of this hyphenated platform, we analyzed a trypsin digested monoclonal antibody (trastuzumab) followed by CESI- MS. At the primary sequence level, 100% sequence coverage of the antibody was achieved. In addition, information about glycosylations and degradation hotspots in the antibody were also obtained in the same CESI-MS run. The CESI 8000 High Performance Separation system together with the high speed and sensitive TripleTOF is an ideal solution for rapid assessment of both peptide sequence coverage and posttranslational modifications of protein biotherapeutics. Introduction Monoclonal antibodies (MAbs) represent a major part of biotherapeutic agents, with 30 MAbs already being in use currently to treat cancer, auto-immune diseases, and systemic infections, and several others in development pipeline 1. Unlike small molecule drugs, MAbs are large proteins with average molecular weight around 150 kda, and also exhibit a high level of heterogeneity owing to glycosylations and other degradative modifications. In addition to the original MAbs, alternative products such as antibody-drug conjugates, biosimilars, biobetters, and next generation antibodies are also being developed to minimize cost and enhance cytotoxicity 2. Consequently, it is important to comprehensively characterize both the originator MAbs at various stages of development and Fig. 1 Schematic representation of the CESI sprayer design production processes, and also the newer alternatives since any changes to the primary amino acid sequence and/or glycosylation pattern will impact the therapeutic efficacy, bioavailability, and biosafety. Currently, a variety of mass spectrometry based methods are employed to characterize MAbs, since MS enables fast, efficient, and thorough analysis necessary to examine these highly complex glycoproteins. Vast majority of the current methods use an up-front separation technique such as liquid chromatography (LC) followed by MS to profile and characterize the MAbs. However, LC based workflows entail separation of the glycosylation part from the polypeptide chain prior to analysis. This deglycosylation step is required to cleave off the hydrophilic glycans which would cause the glycopeptides to elute in the void volume during reversed phase chromatography. On the other hand, although, dedicated stationary phases such as HILIC are available to perform glycan or glycopeptide analyses, it would compromise identification of non-glycosylated peptides. This highlights the need for new technologies in which both hydrophilic glycopeptides and unmodified peptides can be analyzed without additional steps to separate and analyze them independently. Capillary electrophoresis (CE) is based on the migration of solutes inside narrow-bore capillaries under the influence of high electric fields. The most common CE method is capillary zone electrophoresis (CZE) which is based on the differences in the electrophoretic mobilities of the charged species. In addition, electro-osmotic flow (movement of fluid in the capillary under the p 1

2 applied electric field) also causes bulk flow during CZE. Though CE was a prevalent separation technique for more than two decades, it was only in the late 1980 s it was first demonstrated that CE effluent can be interfaced to ESI-MS by using a liquid sheath junction 3. But an inherent problem associated with this technique is that the sheath flow dilutes the analytes migrating from the capillary. A recent advancement to meet the need of interfacing ultra-low flow CE to MS is the introduction of CESI-MS, a combination of CE and ESI by a dynamic process in a single device. The polyimide coating at the capillary outlet is removed and a porous tip is created by etching. The resulting CESI tip is then inserted into an ESI needle and filled with conductive liquid to establish electrical connection (Fig. 1). Using this novel CESI porous tip sprayer design, the analytes are separated based on their electrophoretic mobility, followed by ESI mechanism to introduce the ions into the mass spectrometer to measure the mass/charge ratio of the ions. By integrating CE and ESI into a single device, the inherent benefits of CE such as narrow peak widths, high resolution, and high speed analysis can be coupled with the advantages of ESI. While rapid CE separations reduce analysis time, it also necessitates the use of new generation high speed MS instruments such as the TripleTOF to preserve the separation efficiency. The TripleTOF system used in this work is capable of performing MS and MS/MS at high acquisition speed and at the same time, provides high resolution and high mass accuracy in both modes. We report on the qualitative analysis of biopharmaceuticals using trastuzumab using the on-line connection of a CESI 8000 automated capillary electrophoresis system with a TripleTOF system. Trastuzumab is a humanized recombinant MAb produced in Chinese hamster ovary (CHO) cell lines and is targeted against HER2/neu receptor, which is overexpressed in breast cancer 4. This therapeutic MAb is composed of 2 heavy and 2 light chains with an N-glycosylation at Asn 300. This biopharmaceutical is an ideal representation of IgG1 type MAbs, thus the performance characteristics of the CESI TripleTOF MS platform was evaluated by analyzing the peptides and glycopeptides generated by tryptic digestion. Due to the nature of the CE separation, the glycopeptides were not separated from the unmodified peptides, thus, they were analyzed together during the same run. Experimental Design Sample Preparation: 100 µg of Trastuzumab was solubilized using Rapigest followed by reduction with DTT and alkylation by iodoacetamide. The sample was then digested with trypsin at 37ºC overnight, dried down, and then resuspended in 100 µl of leading electrolyte (100 mm ammonium acetate at ph 4), yielding a final concentration of 1 µg / µl of digested antibody. CESI-MS: CE was performed using the Beckman Coulter CESI 8000 High Performance Separation system sold through SCIEX separations, a part of AB SCIEX. The CESI was connected online with the AB SCIEX TripleTOF Just 50 nl of the sample (equivalent to 100 fmol of digested antibody) in 100 mm leading electrolyte was injected into the bare fused silica separation capillary and transient-isotachophoresis (t-itp) was applied to focus the sample during electrophoretic separation. A 10% acetic acid was used as background electrolyte and a voltage of 20 kv was applied between the inlet vial and the CESI sprayer. Within the separation capillary, the analyte molecules migrate according to their charge/hydrodynamic volume ratio and when they reach the porous sprayer tip of the capillary, the ESI mechanism introduces the ions into the mass spectrometer. Information dependent acquisition (IDA) mode consisting of a high resolution TOF MS survey scan followed by several MS/MS scans was utilized for data acquisition. The IDA parameters are as follows: 250 msec TOF MS survey scan, 50 msec IDA on the top 30 ions which exceed 150 cps, rolling collision energy to induce fragmentation, and the dynamic exclusion time was set to 5 sec. The total cycle time was equal to 1.8 sec and the IDA parameters were optimized so that the duty cycle of the MS is not limiting the high speed CE separation. By enabling autocalibration during CE-MS batches, the instrument was automatically calibrated once every 3 runs, limiting deviation of mass measurement accuracy. Data Analysis: Data analysis was performed using AB SCIEX BioPharmaView Software. Glycopeptides were extracted manually and the MS/MS spectra were checked for diagnostic ions from glycans. Results and Discussion CESI-MS analysis of Trastuzumab: The total ion electropherogram profiles obtained using 100 fmol of trypsin digested trastuzumab run using CESI-MS is illustrated in Fig. 2. The total ion electropherogram is obtained by summing the intensities of all the ions in each spectrum and then plotting the sum as a function of migration time. In the IDA experiment mode, since there are 2 experiments (survey scan and IDA scan), the total ion electropherogram corresponds to the intensity sums of both experiments. p 2

3 Intensity Fig. 2 Total ion electropherograms of trypsin digested trastuzumab run by CESI-MS. Primary Sequence Coverage Analysis: From a single enzymatic digest in a single CESI-MS run, 100% sequence coverage was obtained for both the heavy (HC) and light (LC) chains of the MAb. Peptides ranging from 4 to 63 amino acids in length were detected without any missed cleavages. Electrophoretic separation is based on the charge-to-hydrodynamic volume ratio of the peptides and not dependent on their hydrophobicity. Thus, small hydrophilic peptides, N-terminal Glu which are often lost in the void volume in reversed phase LC, and large hydrophobic peptides, which tend to be retained in the reversed phase column, can be identified equally well resulting in the high sequence coverage obtained by this technique. N-terminal Characterization: The N-terminus of MAbs contains a glutamine or glutamate residue which can undergo cyclization to form pyroglutamate. It is important to characterize this modification A N-terminal pyroglu b 3 pyro EVQLVESGGGLVQPGGSLR B y 6 b 4 b 5 4 y 7 3 y 8 2 b 7 b 9 b 10 b 6 y b 11 5 Fig. 3 (A) Extracted ion electropherogram of N-terminal peptide with Glu and pyroglu separated by CESI and (B) MS/MS identification of N-terminal peptide with pyroglu. p 3

4 since it helps with controlling the antibody production process. The N-terminus of the heavy chain of trastuzumab contains a glutamate residue. Data from CESI-MS analysis showed the presence of both the modified and unmodified forms of this N- terminal peptide (EVQLVESGGGLVQPGGSLR). The coexistence of both the forms indicated that this was a partial modification. Pyroglutamination leads to loss of a positive charge and due to this charge difference, the electrophoretic mobility of the modified peptide is lower than the unmodified one. This is advantageous since the modified and unmodified forms can be separated and detected by CESI-MS. Fig. 3A shows the extracted ion electropherogram of the N-terminal peptide with and without pyroglutamination. Furthermore, the MS/MS spectra acquired from fragmenting the modified peptide confirmed the presence of the pyroglutamate residue (Fig. 3B). Methionine Oxidation: Oxidation of methionine residues is a common modification that occurs during manufacturing and storage of MAbs. The rate of oxidation of different methionine residues in the same MAb is usually different. Also, depending on the location of the susceptible methionine residue, it is important to characterize this modification to ensure its pharmacological properties. Fig. 4 shows the extracted ion electropherograms of the DTLMISR peptide with oxidized and unmodified forms at Met 255 in the HC of trastuzumab. This was found to be a partial modification. Further confirmation of the oxidation at Met 255 by MS/MS is shown in Fig. 5. Another oxidative degradation hotspot at Met 431 residue was also identified using CESI-MS and MS/MS data. Unmodified peptide Oxidized at Met 255 Fig. 4 Extracted ion electropherogram showing peptides with unmodified and oxidized forms at Met 255 in the heavy chain. A S I M(ox) L DTLM (OX) ISR y 3 y 2 b 3 b 4 b 5 S I y 3 M L DTLMISR B y 2 b 3 b 4 Fig. 5 MS/MS identification of peptide with oxidized methionine (A) and unmodified methionine (B) at Met 255 in the heavy chain. b 5 p 4

5 Aspargine Deamidation: Deamidation of aspargine to aspartate/isoaspartate is yet another common modification that can occur in MAbs due to changes in temperature, ph, solvent exposure etc. Deamidation in the antigen binding region of the MAb could lead to potential loss of activity and thus, poses a serious concern. Since there is no charge difference due to the loss of amide group, the electrophoretic mobilities of the deamidated and unmodified peptide are the same, but it can be identified by the difference in mass. Fig. 6 shows the MS/MS identification of deamidation at Asn 55 in the HC. Deamidation at Asn 387 in the HC and at Asn 30 in the LC of trastuzumab were also identified by MS/MS. Glycosylation Heterogeneity: Trastuzumab possesses one N- glycosylation site at Asn 300 in the HC where different glycoforms including a-fucosylated or fucosylated complex glycans can be present. Since there are two heavy chains in the mab, this gives rise to different glycosylation combinations that can be attached presenting significant complexity for the analysis. Though techniques to release glycans are widely used, the site of attachment of the glycan moiety to the specific amino acid residue can be identified only by methods that do not require deglycosylation. By using CESI-MS, the G0F, G1F, and G2F forms of the modified peptide TKPREEQYNSTYR can be separated well due to the migration time difference between these forms as shown in Fig. 7. Furthermore, the identification of G0F, G1F, and G2F forms of the peptide EEQYNSTYR without the missed cleavage also confirmed the presence of these glycoforms. In addition, the afucosylated forms of this peptide, such as G0 and G1, were identified, but it has to be further confirmed that the afucosylated forms were not generated due to source fragmentation of fucosylated counterparts. Though CAD is known for yielding less backbone cleavage, it serves to provide information about the diagnostic ions in the glycan and can be employed for routine analysis of the antibody. Unmodified peptide N T P Y y 7 A y 6 y Deamidated at Asn 55 N* T P Y y 7 B y 6 y Fig. 6 MS/MS identification of deamidation with (A) showing unmodified peptide and (B) deamidation at Asn 55 in the heavy chain. p 5

6 G0F G1F G2F Fig. 7 Extracted ion electropherograms of the peptide TKPREEQYNSTYR with G0F, G1F, and G2F modifications. Conclusions We have presented a new platform combining CESI, a robust and highly efficient separation and electrospray technique connected on-line with a TripleTOF MS, a high resolution accurate mass measurement system for qualitative analysis of biopharmaceuticals. In this new CESI design, after electrophoresis, the analyte molecules were electrosprayed directly into the mass spectrometer by making the terminal end of the separation capillary a sprayer. The droplets formed during the electrospray process in the CESI design are small and this results in more efficient ionization. With flow rates as low as 25 nl/min, the ion suppression was minimized thus providing higher sensitivity to analyze less abundant glycopeptides present in the same sample. Furthermore, this also revealed PTM hotspots in the antibody such as methionine oxidations, deamidations, and cyclization of N-terminal glutamate to pyroglutamate. In addition, sample consumption is minimized since only very low amounts of samples are injected. Nevertheless, preconcentration techniques can be performed if larger amounts have to be injected. The high throughput CESI-MS analysis of the antibody products that we have presented here is attractive for workflows where rapid verification of the primary sequence and glycopeptide profiling are required. The combination of high separation efficiency and high sensitivity allows the efficient analysis of all peptides including modified and low abundant species, in addition to confirming the amino acid sequence of the antibody. References 1. Wagner-Rousset, E., Schaeffer-Reiss, C., Bednarczyk, A., Corvaia, N., Van Dorsselaer, A., Beck, A. Methods in Molecular Biology 2013, 988, Beck, A., Sanglier-Cianferani, S., Van Dorsselaer, A. Analytical chemistry 2012, 84, Smith, R., Udseth, H. Nature 1988, 331, Damen, C., Chen, W., Chakraborty, A., van Oosterhout, M., Mazzeo, J., Gebler, J., Schellens, J., Rosing, H., Beijnen, J. Journal of American Society of Mass Spectrometry 2009, 20, For Research Use Only. Not for use in diagnostic procedures AB SCIEX. SCIEX is part of AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Publication number: p 6