Anal. Chem. 2009, 81, 1686–1692
Direct Identification and Quantification of Aspartyl Succinimide in an IgG2 mAb by RapiGest Assisted Digestion Holly Z. Huang,*,† Andrew Nichols,‡ and Dingjiang Liu† Analytical and Formulation Sciences and Formulation and Analytical Resources, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320 A special tryptic digestion method has been developed to facilitate rapid identification and accurate quantification of site-specific aspartyl succinimide (Asu) formation in complex protein molecules, such as monoclonal antibodies (mAbs). This method replaces chaotropic reagents, such as urea and guanidine hydrochloride (GdnHCl) with an acid labile surfactant RapiGest (RG), eliminates alkylation and desalting steps, and accomplishes the reduced tryptic digestion of an IgG2 mAb in a mildly acidic condition (pH 6.0) with half the time required by conventional methods. The new digestion condition preserves the labile Asu during sample preparation and solves the problem that conventional method has been facing in detecting and quantifying Asu in complex proteins. The validity of this method was confirmed by subjecting a mixture of peptides containing a predetermined amount of Asu to the same digestion conditions. An excellent correlation was also observed for the Asu results from cation-exchange chromatography (CEX) and tryptic peptide maps generated with the new digestion method. This method is also applicable to other enzymatic digestions and used to monitor site-specific deamidation, isomerization, and other chemical modifications in complex proteins by LC/MS. Isomerization and deamidation are major non-enzymatic posttranslational modifications of proteins under typical formulation and storage conditions (pH 4.5-7.5, 2-37 °C).1,2 In proteins and peptides, Asn, Asp residues (Asx) are the most susceptible amino acid residues to deamidation and isomerization, respectively. The degradation of Asx in proteins can lead to loss of in vivo biological functions or in vitro stability, and raise safety and efficacy concerns of biological therapeutics.3-8 In addition, the molecular heterogeneity due to these modifications may create challenges to * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: (805)-447-4670. Fax: (805)-376-8505. † Analytical and Formulation Sciences. ‡ Formulation and Analytical Resources. (1) Clarke, S.; Stephenson, R. C.; Lowenson, J. D. In Stability of Protein Pharmaceuticals, Part A: Chemical and Physical Pathways of Protein Degradation; Ahern, T. J., Manning, M. C., Eds.; Plenum Press: New York, 1992, pp 1-29. (2) Powell, M. F. In Formulation, Chracterization, and Stability of Protein Drugs; Pearlman, R., Wang, Y. J., Eds.; Plenum Press: New York, 1996, pp 1140. (3) Flatmark, T.; Sletten, K. J. Biol. Chem. 1968, 243, 1623–1629.
1686
Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
manufacturing consistent products. Therefore, it is essential to have a reliable and effective analytical method to characterize and monitor the degradation products of Asx in proteins and protein therapeutics during different development processes and stages. Early studies with peptides suggest that deamidation and isomerization reactions proceed through a succinimide intermediate before converting to isoAsp and Asp (Scheme 1).9-11 Depending on solution pH, primary amino acid sequence and protein conformation, succinimide can also accumulate in proteins and become the degradation end products (IsoAps and Asp) under proper conditions.5,12-16 However, characterization of succinimide formation in large protein molecules such as monoclonal antibody is more challenging than that of small peptides because of the instability of aspartyl succinimide (Asu) in neutral and alkaline solutions, the structural complexity of large protein molecules, and the limitation of available analytical methods. Commonly used methods for Asu detection in proteins include but are not limited to enzymatic methylation of isoaspartate by D-aspartyl/L-isoaspartyl methyltransferase17 or protein L-isoaspartyl methyltransferase (PIMT),18 followed by radioactive detection or HPLC separation; chemical hydrolysis with hydroxylamine19-21 and chromatography (4) Harding, J. J.; Beswick, H. T.; Ajiboye, R.; Huby, R.; Blakytny, R.; Rixon, K. C. Mech. Ageing Dev. 1989, 50, 7–16. (5) Cacia, J.; Keck, R.; Presta, L. G.; Frenz, J. Biochemistry 1996, 35, 1897– 1903. (6) Harris, R. J.; Kabakoff, B.; Macchi, F. D.; Shen, F. J.; Kwong, M.; Andya, J. D.; Shire, S. J.; Bjork, N.; Totpal, K.; Chen, A. B. J. Chromatogr., B: Biomed. Sci. Appl. 2001, 752, 233–245. (7) Paborji, M.; Pochopin, N. L.; Coppola, W. P.; Bogardus, J. B. Pharm. Res. 1994, 11, 764–771. (8) Kroon, D. J.; Baldwin-Ferro, A.; Lalan, P. Pharm. Res. 1992, 9, 1386–1393. (9) Swallow, D. L.; Abraham, E. P. Biochem. J. 1958, 70, 364–373. (10) Clarke, S. Int. J. Pept. Protein Res. 1987, 30, 808–821. (11) Geiger, T.; Clarke, S. J. Biol. Chem. 1987, 262, 785–794. (12) Teshima, G.; Stults, J. T.; Ling, V.; Canova-Davis, E. J. Biol. Chem. 1991, 266, 13544–13547. (13) Violand, B. N.; Schlittler, M. R.; Kolodziej, E. W.; Toren, P. C.; Cabonce, M. A.; Siegel, N. R.; Duffin, K. L.; Zobel, J. F.; Smith, C. E.; Tou, J. S. Protein Sci. 1992, 1, 1634–1641. (14) Tomizawa, H.; Yamada, H.; Ueda, T.; Imoto, T. Biochemistry 1994, 33, 8770–8774. (15) Markell, D.; Hui, J.; Narhi, L.; Lau, D.; LeBel, C.; Aparisio, D.; Lile, J.; Jing, S.; Yui, D.; Chang, B. S. Pharm. Res. 2001, 18, 1361–1366. (16) Chu, G. C.; Chelius, D.; Xiao, G.; Khor, H. K.; Coulibaly, S.; Bondarenko, P. V. Pharm. Res. 2007, 24, 1145–1156. (17) Ota, I. M.; Ding, L.; Clarke, S. J. Biol. Chem. 1987, 262, 8522–8531. (18) Potter, S. M.; Henzel, W. J.; Aswad, D. W. Protein Sci. 1993, 2, 1648– 1663. (19) Kwong, M. Y.; Harris, R. J. Protein Sci. 1994, 3, 147–149. (20) Zhu, J. X.; Aswad, D. W. Anal. Biochem. 2007, 364, 1–7. 10.1021/ac802708s CCC: $40.75 2009 American Chemical Society Published on Web 01/15/2009
Scheme 1. Schematic Diagram for Asp/Asn Degradations via Asu Intermediate
separations (IEX or HIC) of intact molecule, followed by fraction collection, N-terminal sequencing, and tryptic digestions.5,12,16 Although these methods can detect succinimide in proteins, the evidence for succinimide is often indirect. In addition, the procedures are usually lengthy and tedious. Mass spectrometry provides powerful alternative solutions to characterize Asx modifications. Tandem mass spectrometry (MS/ MS) allows detection and quantification of Asx degradation products with site specific information. Electron captured dissociation (ECD), electronic transfer dissociation (ETD),22-25 as well as collisionally activated dissociation (CAD) MS/MS have been used for isoAsp and Asp identifications and differentiations.26-28 Applications of 18O isotope labeling along with tryptic digestion to quantify Asu in a mAb29 and differentiate deamidation artifact in proteins30 were also reported. Recent advancement in high resolution mass spectrometry has made possible the measurement of Asn deamidation in intact proteins with mass defect and isotopic envelop deconvolution methods using Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS).31-34 It was reported that no sample preparation was required for the envelop deconvolution method using FTMS for molecules of about 200 residues.33 However, for large (21) Alfaro, J. F.; Gillies, L. A.; Sun, H. G.; Dai, S.; Zang, T.; Klaene, J. J.; Kim, B. J.; Lowenson, J. D.; Clarke, S. G.; Karger, B. L.; Zhou, Z. S. Anal. Chem. 2008, 80, 3882–3889. (22) Cournoyer, J. J.; Pittman, J. L.; Ivleva, V. B.; Fallows, E.; Waskell, L.; Costello, C. E.; O’Connor, P. B. Protein Sci. 2005, 14, 452–463. (23) Cournoyer, J. J.; Lin, C.; O’Connor, P. B. Anal. Chem. 2006, 78, 1264– 1271. (24) O’Connor, P. B.; Cournoyer, J. J.; Pitteri, S. J.; Chrisman, P. A.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2006, 17, 15–19. (25) Cournoyer, J. J.; Lin, C.; Bowman, M. J.; O’Connor, P. B. J. Am. Soc. Mass Spectrom. 2007, 18, 48–56. (26) Carr, S. A.; Hemling, M. E.; Bean, M. F.; Roberts, G. D. Anal. Chem. 1991, 63, 2802–2824. (27) Castet, S.; Enjalbal, C.; Fulcrand, P.; Guichou, J. F.; Martinez, J.; Aubagnac, J.-L. Rapid Commun. Mass Spectrom. 1996, 10, 1934–1938. (28) Gonzalez, L. J.; Shimizu, T.; Satomi, Y.; Betancourt, L.; Besada, V.; Padron, G.; Orlando, R.; Shirasawa, T.; Shimonishi, Y.; Takao, T. Rapid Commun. Mass Spectrom. 2000, 14, 2092–2102. (29) Xiao, G.; Bondarenko, P. V.; Jacob, J.; Chu, G. C.; Chelius, D. Anal. Chem. 2007, 79, 2714–2721. (30) Li, X.; Cournoyer, J. J.; Lin, C.; O’Connor, P. B. J. Am. Soc. Mass Spectrom. 2008, 19, 855–864. (31) Schmid, D. G.; von der Mulbe, F. D.; Fleckenstein, B.; Weinschenk, T.; Jung, G. Anal. Chem. 2001, 73, 6008–6013. (32) Robinson, N. E.; Lampi, K. J.; McIver, R. T.; Williams, R. H.; Muster, W. C.; Kruppa, G.; Robinson, A. B. Mol. Vision 2005, 11, 1211–1219. (33) Zabrouskov, V.; Han, X.; Welker, E.; Zhai, H.; Lin, C.; van Wijk, K. J.; Scheraga, H. A.; McLafferty, F. W. Biochemistry 2006, 45, 987–992. (34) Robinson, N. E.; Zabrouskov, V.; Zhang, J.; Lampi, K. J.; Robinson, A. B. Rapid Commun. Mass Spectrom. 2006, 20, 3535–3541.
proteins such as mAbs (150 kDa, >600 residues and 18 pairs of disulfide bonds), bottom-up LC/MS analysis is often the method of choice for monitoring site specific modifications, generally by trypsin digestions to generate smaller peptides to facilitate MS detections. Conventional tryptic digestions are typically performed at pH between 7.5 and 8.5 to maximize trypsin activity. However, theses conditions are not suitable for analyzing succinimide as it will rapidly hydrolyze and convert to Asp and isoAsp. Furthermore, these conditions may also promote method induced deamidation, which can mislead data interpretation.30 Because the stability of Asu is highly pH dependent, it is possible to quantify Asu directly from tryptic peptide maps at a lower pH condition to maintain its stability during sample preparation. On the other hand, trypsin will be less efficient at acidic pH, it will require higher trypsin to protein ratio, and longer digestion time to compensate for the loss of efficiency. In this case, trypsin autolysis and acid hydrolysis due to extended incubation under acidic conditions may occur, which can complicate data analysis and result interpretation. To overcome the above problems and develop an efficient tryptic digestion method under less favorable (acidic pH) conditions, we replaced the traditional chaotropic reagent such as Guanidine HCl (GdnHCl) and urea in the denaturation step with RapiGest (RG), an acid labile surfactant, which decomposes to two small molecules under strong acidic conditions, one of which will precipitate out of solution, and the other one is soluble and does not interfere with chromatographic separations.35 A previously characterized IgG2 mAb and known for Asu accumulation at aspartic acid residue position 30 (Asp30) was used as a model protein for the method development.16 EXPERIMENTAL SECTION Materials. The recombinant human IgG2 mAb was expressed in CHO cells and purified by Amgen (Seattle, WA). The IgG2 mAb consists of 661 amino acid residues with 18 disulfide bonds and has a molecular weight of about 150 kDa. The degraded IgG2 sample was obtained by formulating the IgG2 at pH 5.2 and incubating for 1 year at 29 °C. A hexadecapeptide (N96) VVSVLTVLHQDWLNGK from the Fc region of an IgG1 mAb was purchased from SynPep (Dublin, CA). The peptide was dissolved into water to make a 10 mg/ mL stock solution. To generate deamidated peptide, the stock (35) Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S.; Gebler, J. C. Anal. Chem. 2003, 75, 6023–6028.
Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
1687
Figure 1. RP-HPLC separation overlay of the tryptic digests of the IgG2. UV signals at 214 nm were used for detection. Different amounts of the IgG2 were incubated with 0.1% RG at 60 °C for 30 min. DTT and IAM were added proportionally to the amount of protein. Trypsin was added at a 1:50 (w/w) ratio to the protein and incubated at 37 °C for 3.5 h. TFA was added last to quench digestion and decompose RG.
Figure 2. Tryptic maps with and without alkylation at pH 6.5. The circle highlights multiple peaks with 57 Da increment, suggesting incomplete alkylation.
solution was diluted to 0.1 mg/mL with 10 mM sodium phosphate buffer at pH 7.0, and incubated for 2 days at 37 °C. Chemical Reagents. Lyophilized RapiGestSF was obtained from Waters (Milford, MA). Tris (2-carboxyethyl)phosphine (TCEP), trifluoroacidic acid (TFA), and Zeba micro desalt spin columns were purchased from Pierce (Rockford, IL). Dithiotreitol (DTT) was obtained from Roche (Indianapolis, IN), iodoacetamide (IAM) was from Sigma (St Louise, MO). Sequencing 1688
Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
grade modified and TPCK treated trypsin was purchased from Promega (Madison, WI). Conventional Tryptic Digestion Method. The mAb was denatured with 8 M GdnHCl at pH 7.5 and reduced by 0.5 M DTT, followed by alkylation with 0.5 M IAM. Desalting and buffer exchange were accomplished by Zeba desalt spin columns. Trypsin digestion was completed in 5 h at 37 °C with an enzyme to protein ratio of 1:10 (w/w).
Figure 3. Effect of pH and digestion time on peak 1-3. Peak 1 (L3_isoAsp30) as indicated by a solid gray line, has the same mass of 1496.6 Da as peak 2 (L3_Asp30); peak 3 (L3_Asu30) as designated by a dotted-line, has a mass of 1478.6 Da. The stressed IgG2 sample was treated with 0.1% RG at pH 5.9 and then reduced with TCEP at 60 °C, followed by dilution with digestion buffer of pH 6.8, 7.5, and 8.5, respectively.
Tryptic Peptide Separation by RP-HPLC and MS/MS Analysis. A Polaris C18 Ether column (3 µm, 2.1 × 250 mm, 300 Å, Varian, CA) was used to separate the digested IgG2 peptides. The column temperature was set at 50 °C with a flow rate of 0.2 mL/min. Mobile phase A was 0.1% aqueous TFA, and mobile phase B was 90% acetonitrile with 0.085% aqueous TFA. A linear gradient ramped up from 0 to 50% B over 185.5 min. A Thermo Fisher Scientific LCQ Deca ion trap mass spectrometer was connected in-line with an Agilent 1100 HPLC system to identify the eluting peptides. The mass spectra of the peptides and their fragments were obtained by a triple play method, which included a full scan, followed by zoom and MS/ MS scans. A standard off-axis ESI source was used as an atmosphere-vacuum interface. The spray voltage was 5 kV, and the capillary temperature was 250 °C. The MS/MS spectra were obtained using ion trap collision energy of 35%. RESULTS AND DISCUSSION Tryptic Digestion with RG. Secondary and tertiary structural analyses of the IgG2 by far UV CD and FL spectroscopy suggest that 0.1% RG acts as a mild denaturant to perturb rather than completely unfold the IgG2 structures (Supporting Information, Figure S1-2). One important aspect of a successful RG assisted digestion (RAD) is the effective IgG2 to RG ratio. Four different IgG2 to RG ratios were compared at pH 6.5, while RG was kept constant at 0.1% (w/v). As the amount of IgG2 decreased from 300 µg to 50 µg, improved recoveries for early eluting peaks were observed, as highlighted by the rectangle
in Figure 1, indicating more effective digestions can be achieved at higher RG to IgG2 ratios. But when the IgG2 was below 50 µg, the final concentration of the digest was too low for LC/ MS analysis. Therefore, the working range for the IgG2 was chosen from 50 to 100 µg in 0.1% RG solution. Typically, dithiothreitol (DTT) is used as a reducing agent in the denaturing buffer (pH g 7) in conventional tryptic digestion protocols. However, DTT becomes less effective in acidic digestion buffers (pH < 7) as thiolate, and the reactive form of DTT decreases with pH owing to its high pKa value of 8.3. We solved this problem by replacing DTT with tris (2carboxyethyl)phosphine (TCEP), which is stable at a wide pH range and capable of reducing disulfide bonds specifically and completely under acidic conditions.36,37 In conventional tryptic digestion protocols, iodoacetamide (IAM) is used to alkylate free thiol groups after the reduction step to prevent disulfide bonds from reforming or scrambling at basic pH conditions, otherwise, misfolding and aggregation could occur after the removal of chaotropic reagents (desalting) prior to the digestion. However, under mildly acidic conditions, the majority of reduced disulfide bonds no longer exist as thiolate because of their high pKa values (8.5-10.0 depending on adjacent amino acids). For example, at pH 6.0, only a very small fraction (∼0.5%) of cysteine exists as thiolate, which is the reactive form of cysteine that enables the (36) Levison, M. E.; Josephson, A. S.; Kirschenbaum, D. M. Experientia 1969, 25, 126–127. (37) Han, J. C.; Han, G. Y. Anal. Biochem. 1994, 220, 5–10.
Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
1689
Figure 4. Tandem mass spectra of the tryptic peptide L3 derived by CID of the doubly charged precursor ions (M + 2H)2+. (a) Peak 2, m/z ) 749.15 Da; and (b) peak 3, m/z ) 740.15 Da. The assignments of y- and b-ions were labeled on the top and bottom of the peptide sequences, respectively.
alkylation reaction, making it extremely difficult to carry out the alkylation reaction. To solve this problem, we added an excess amount of TCEP and kept it in the digestion buffer without removal. As a result, our reduced tryptic digestions at pH 6.5 yielded a peptide map that was free of alkylated cysteines and non-native disulfide bonds (Figure 2, bottom trace). In comparison, the reduced and alkylated tryptic map of the IgG2 at the same pH detected multiple peptide peaks with 57 Da mass increments, indicating incomplete alkylation 1690
Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
reactions (Figure 2, top trace). The two peptide maps look somewhat different; this is due to the hydrophobicity change of the thiol containing peptides after alkylation. Hence, the retention time shifted as compared to the non-alkylated peptide map. To accurately quantify Asu in the IgG2 from a tryptic map, it is essential to identify the right pH conditions for trypsin digestions. First, the individual peaks associated with Asp degradation need to be identified. This was accomplished by varying
Table 1. Effect of Each Digestion Step on N96 Peptide procedure
time/ temperature
%IsoD96 %N96 %D96 %Asu96
t)0 denaturation/reduction 30 min, 60 °C digestion 2 h, 37 °C quenching/remove Rg 30 min, 37 °C (pH 2)
22.3 22.2 22.1 20.4
64.8 9.5 65.9 8.8 66.2 8.7 65.5 10.3
3.5 3.0 3.0 3.8
Table 2. CEX and Tryptic Map Results for Asu in Different Formulations at 29°C, 1 year formulation pH 30
Figure 5. pH and time dependent inter conversion of Asp30, isoAsp30, and Asu30 in the tryptic L3 peptide. The same sample as in Figure 3 was diluted into 0.1% RG buffers at pH 6.0, 6.5, and 6.9 correspondingly, and then denatured and reduced with TCEP at 60 °C for 30 min, followed by trypsin digestions at 37 °C for 1-4 h.
the pH of the digestion buffer between 6.8 and 8.5, as well as the time for trypsin digestion between 2 and 4 h. Figure 3 shows clear evidence of pH and time dependence of peaks 1-3 (labeled on the bottom trace of Figure 3) for the stressed sample (pH 5.2, one year storage at 29 °C). As the digestion buffer pH increased from 6.8 to 8.5, peak 3 decreased dramatically, and it even disappeared at pH 8.5. In contrast, peak 1 was growing rapidly. At the same pH, peak 3 also decreased with extended incubation time, accompanied with an increase in peak 1, except for pH 8.5, because there was no peak 3 to begin with for detection. In the frozen samples (-70 °C), peak 2 was the only main component with a minimum of peak 3 (data not shown). LC/MS analysis of peak 1 and 2 showed the same mass of 1496.6 Da; peak 3 detected a mass of 1478.6 Da, which is 18 Da lighter than peak 1 and 2, matching the loss of one water molecule. A database search for the IgG2 amino acid sequence found that the mass of 1496.6 Da matched the light chain sequence of 25-ASQSVD30SNLAWYR37 and the mass of 1478.6 Da coincided with the conversion of Asp30 to Asu30. MS/MS analyses were carried out for peak 2 and 3. Their MS/MS spectra derived from collision-induced dissociation (CID) of the (M + 2H)2+ precursor ions are compared in Figure 4. On the basis of the characteristics of the interconversions of peak 1-3, their mass values and MS/MS results, it can be concluded that peak 2 is the native tryptic peptide L3_Asp30, with the sequence 25-ASQSVD30SNLAWYR-37; peak 1 is the isomerization product of peak 2 with the sequence 25ASQSVisoD30SNLAWYR-37, in which Asp30 converts to IsoAsp30 via Asu30, and peak 3 contains the Asu intermediate with the sequence 25-ASQSVAsu30SNLAWYR-37. As demonstrated in Figure 5, it is very difficult to capture the Asu formation with digestion conditions at pH > 7, even within 2 h at 37 °C. Tryptic digestions with RG in a narrow pH range from 6.0 to 6.9 were compared to define the optimal condition for Asu quantification. Figure 5 summarizes the impact of pH and incubation time on Asu detection during digestion. At a specific pH, %Asp30 showed little change over time (
