Characterization of Antibody Charge Heterogeneity Resolved by

Anal. Chem. 2010, 82, 3510–3518

Characterization of Antibody Charge Heterogeneity Resolved by Preparative Immobilized pH Gradients Charlie D. Meert, Lowell J. Brady, Amy Guo, and Alain Balland* Amgen Inc., Analytical and Formulation Sciences, 1201 Amgen Court West, Seattle, Washington 98119 A capillary isoelectric focusing (cIEF) method has been developed as an alternative to cation exchange chromatography to determine charge heterogeneity for a therapeutic antibody. Characterization of the cIEF profile is important to understand the charged isoform distribution. A variety of preparative IEF methods have been developed over the years but have had various limitations including high levels of contaminating ampholytes and complex fractionation and isolation procedures. More recently, an off-line method that uses pI-based separation on immobilized pH gradients was developed to preparatively isolate material with convenient liquid phase recovery. This method uses the Agilent OFFGEL 3100 Fractionator and was optimized to produce fractions of antibody charge isoforms differing by as little as 0.1 pI units. The isolation of highly resolved fractions then allowed for the identification of N- and C-terminal basic charge modifications including noncyclized glutamine, signal peptide extensions, and various levels of C-terminal lysine processing and high mannose structures. These species could then be correlated to specific peaks in the cIEF profile. This work shows that a preparative IEF method using immobilized pH gradients can be optimized to generate highly resolved, pI-based fractions in solution which can be used for successful cIEF profile characterization. Access to preparative amounts of discrete charged species allows for a better understanding of the underlying covalent modifications responsible for the charge differences and facilitates evaluation of the impact of these modifications on stability and potency of therapeutic antibodies. In the area of therapeutic monoclonal antibody production and characterization, assays that separate antibody populations by their charge are essential for defining the purity of the final molecule, detecting post-translational and chemical modifications, and assessing stability. Defining charge heterogeneity at a molecular level can help explain potential variations in potency among product isoforms and provide an increased understanding of degradation pathways. Due to the common occurrence of charge modifications, regulatory agencies usually require that a chargebased assay be included in filings and used for product release to ensure lot-to-lot consistency and stability. Antibody charge heterogeneity is frequently determined by liquid chromatography * Corresponding author. Phone: 206-265-8603. Fax: 206-217-4692. E-mail: [email protected].

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methods and, in particular, cation exchange chromatography (CEX). Characterization of CEX profiles by peak collection and orthogonal analyses, often using mass spectrometry (MS), has enabled the identification of many post-translational and chemical modifications responsible for antibody charge heterogeneity.1 These modifications include asparagine deamidation, stable succinimide formation, C-terminal lysine processing, N-terminal glutamine cyclization, and O-fucosylation.2-6 While CEX remains highly useful for distinguishing chargebased heterogeneities, challenges such as variability between column lots, column fouling, and lack of resolution and robustness for routine characterization of certain antibodies highlight the need for alternate methods for charge-based analysis. Capillary isoelectric focusing (cIEF) is one such technique that continues to gain use and acceptance for characterization of charge heterogeneity and experimental pI determination.7-12 While CEX separates populations of antibody primarily based on the surface charge of solvent-exposed residues, cIEF is a highly resolving technique that separates species primarily based on the pI, or intrinsic net charge, of the molecule and takes into account surface-exposed as well as internal amino acids. This is accomplished by focusing the isoforms in a pH gradient through the application of an electric field, followed by chemical mobilization and UV detection. Due to the difference in separation principles and the exquisite resolution that can be achieved, cIEF offers both a complementary technique and an attractive alternative to ion exchange chromatography. A challenge in the use of cIEF as an analytical technique is the characterization of the resulting antibody cIEF profile. Two (1) Vlasak, J.; Ionescu, R. Curr. Pharm. Biotechnol. 2008, 9, 468–481. (2) Zhang, W.; Czupryn, M. J. J. Pharm. Biomed. Anal. 2003, 30, 1479–1490. (3) Yan, B.; Steen, S.; Hambly, D.; Valliere-Douglass, J.; Bos, T. V.; Smallwood, S.; Yates, Z.; Arroll, T.; Han, Y.; Gadgil, H.; Latypov, R. F.; Wallace, A.; Lim, A.; Kleemann, G. R.; Wang, W.; Balland, A. J. Pharm. Sci. 2009, 98, 3509–3521. (4) Harris, R. J. J. Chromatogr., A 1995, 705, 129–134. (5) Valliere-Douglass, J. F.; Brady, L. J.; Farnsworth, C.; Pace, D.; Balland, A.; Wallace, A.; Wang, W.; Treuheit, M. J.; Yan, B. Glycobiology 2009, 19, 144– 152. (6) Moorhouse, K. G.; Nashabeh, W.; Deveney, J.; Bjork, N. S.; Mulkerrin, M. G.; Ryskamp, T. J. Pharm. Biomed. Anal. 1997, 16, 593–603. (7) Dolnik, V. Electrophoresis 2008, 29, 143–156. (8) Hunt, G.; Moorhouse, K. G.; Chen, A. B. J. Chromatogr., A 1996, 744, 295–301. (9) Hunt, G.; Hotaling, T.; Chen, A. B. J. Chromatogr., A 1998, 800, 355–367. (10) Lasdun, A. M.; Kurumbail, R. R.; Leimgruber, N. K.; Rathore, A. S. J. Chromatogr., A 2001, 917, 147–158. (11) Ma, S. Dev. Biol.(Basel) 2005, 122, 49–68. (12) Silvertand, L. H.; Torano, J. S.; van Bennekom, W. P.; de Jong, G. J. J. Chromatogr., A 2008, 1204, 157–170. 10.1021/ac902408r  2010 American Chemical Society Published on Web 04/05/2010

ways of approaching characterization are the coupling of online characterization techniques such as mass spectrometry and the off-line preparative scale fractionation of material for further characterization. Though progress has been made in the field of cIEF-MS for antibody characterization,13 there remains the difficulty of maintaining optimal separation concurrently with optimal MS detection. This is due to suppression of sample ionization by the low mass ampholytic compounds which form the pH gradient needed for optimal separation and the need to avoid components in the sample preparation that are incompatible with mass spectrometry. Other coupled techniques, such as cIEF-MALDITOF-MS involving a plate spotting step,14 and the addition of an RP-HPLC system between the cIEF and mass spectrometer,15 have improved performance, though obtaining sufficient sensitivity remains a challenge.12 In the area of off-line cIEF characterization, alternate techniques, such as CEX or boronate affinity, followed by reinjection on cIEF, have been employed.16,17 However, this often yields suboptimal fraction purity due to differences in separation principles, making it difficult to determine the identity of individual peaks in the cIEF profile. While these approaches can provide valuable, though often limited information, the need still remains for an easy-to-use, preparative scale technique that exploits isoelectric point as a separation principle. Obtaining nativestate material fractionated by isoelectric point would allow for the determination of other key characteristics such as therapeutic potency and enable accurate characterization of cIEF profiles and charge isoforms differing by isoelectric point. Analytical cIEF relies on small diameter capillaries in which submicrogram quantities of analyte are separated in each run. This makes the use of these methods impractical for preparative fractionation. A number of preparative isoelectric focusing separations of proteins and peptides in solution have been developed to address this problem and many have resulted in successful separations.18 Isoelectric focusing in solution as a preparative technique was first proposed by Bier19,20 and led to the subsequent development of the Rotofor, an instrument commercialized by BioRad. The Rotofor contains 20 compartments and relies on the addition of carrier ampholytes to form the pH gradient used for separation. Successful separations of complex mixtures of proteins and peptides in the presence of a carrier ampholyte solution using the Rotofor have been published.21,22 To avoid the use of ampholytes, Righetti and collaborators proposed the concept of preparative isoelectric focusing using immobilized pH gradients in a six-compartment electrolyzer with each chamber separated (13) Wehr, T. LCGC North Am. 2009, 22, 998–1004. (14) Silvertand, L. H.; Torano, J. S.; de Jong, G. J.; van Bennekom, W. P. Electrophoresis 2009, 30, 1828–1835. (15) Zhou, F.; Hanson, T. E.; Johnston, M. V. Anal. Chem. 2007, 79, 7145– 7153. (16) Quan, C.; Alcala, E.; Petkovska, I.; Matthews, D.; Canova-Davis, E.; Taticek, R.; Ma, S. Anal. Biochem. 2008, 373, 179–191. (17) Sosic, Z.; Houde, D.; Blum, A.; Carlage, T.; Lyubarskaya, Y. Electrophoresis 2008, 29, 4368–4376. (18) Righetti, P. G.; Castagna, A.; Herbert, B.; Reymond, F.; Rossier, J. S. Proteomics 2003, 3, 1397–1407. (19) Bier, M.; Long, T. J. Chromatogr. 1992, 604, 73–83. (20) Bier, M. Electrophoresis 1998, 19, 1057–1063. (21) Brobey, R. K.; Soong, L. Proteomics 2007, 7, 116–120. (22) Velasquez, E. V.; Creus, S.; Trigo, R. V.; Cigorraga, S. B.; Pellizzari, E. H.; Croxatto, H. B.; Campo, S. Hum. Reprod. 2006, 21, 916–923.

by isoelectric membranes prepared at a specific pH.23 Proteins are isolated in the liquid phase between two membranes that bracket their pI. Hoeffer-Pharmacia developed a commercial instrument, the Isoprime, based on this concept. Using this technique, we and others have reported the efficient fractionation of closely related protein isoforms.24,25 Several alterations of the concept of preparative IEF in liquid phase have also been proposed, for example, using a continuous free-flow electrophoresis device,26,27 as well as various modifications of instrumental design, including the Gradiflow apparatus28,29 and the vortexstabilized electrophoresis device.30,31 An alternative instrument for preparative isoelectric focusing with liquid phase recovery is the OFFGEL 3100, introduced by Agilent in 2006.32 This technique represents a variation on preparative isolation using immobilized pH gradient (IPG) gel strips as described by Cargile et al.33 The OFFGEL instrument uses IPG gel strips in a tray underneath a 12 or 24 well attachment that snaps in place over the strips. The aqueous starting sample containing protein and low levels of ampholytes is loaded across all of the wells, and because there is no fluid connection between the wells, the protein migrates through the IPG gel to its pI when voltage is applied. Equilibrium is formed between protein in the gel and in the aqueous phase. After focusing, the protein present in the liquid phase is conveniently recovered from the wells, exchanged into buffer, and concentrated prior to further analysis. This instrument offers several advantages over other methods of isolation, including the potential for high resolution of charged species due to the use of IPG gel strips and a 24 well design, and the ability to use low levels of ampholyte and multiple fractionation conditions in a single run. The instrument is also easy to use and is commercially available and supported by the manufacturer. While the OFFGEL technique has thus far been used for separation of complex peptide and protein mixtures for proteomic applications,34-36 in this work, we apply it to the fractionation of the charge isoforms of a purified therapeutic monoclonal antibody. Using the same separation principle as cIEF, the OFFGEL system allowed for the separation of isoforms differing by as little as 0.1 pI units. This was particularly useful for the antibody of interest since CEX chromatography did not deliver a robust or highly resolving separation. Characterization of the fractions was then successfully pursued using various analytical techniques, including (23) Righetti, P. G.; Wenisch, E.; Faupel, M. J. Chromatogr. 1989, 475, 293– 309. (24) Balland, A.; Mahan-Boyce, J. A.; Krasts, D. A.; Daniels, M.; Wang, W.; Gombotz, W. R. J. Chromatogr., A 1999, 846, 143–156. (25) Zhu, Y.; Lubman, D. M. Electrophoresis 2004, 25, 949–958. (26) Moritz, R. L.; Ji, H.; Schutz, F.; Connolly, L. M.; Kapp, E. A.; Speed, T. P.; Simpson, R. J. Anal. Chem. 2004, 76, 4811–4824. (27) Xie, H.; Bandhakavi, S.; Griffin, T. J. Anal. Chem. 2005, 77, 3198–3207. (28) Horvath, Z. S.; Corthals, G. L.; Wrigley, C. W.; Margolis, J. Electrophoresis 1994, 15, 968–971. (29) Ogle, D.; Sheehan, M.; Rumbel, B.; Gibson, T.; Rylatt, D. B. J. Chromatogr., A 2003, 989, 65–72. (30) Tracy, N. I.; Ivory, C. F. Electrophoresis 2004, 25, 1748–1757. (31) Tracy, N. I.; Ivory, C. F. Electrophoresis 2008, 29, 2820–2827. (32) Horth, P.; Miller, C. A.; Preckel, T.; Wenz, C. Mol. Cell Proteomics 2006, 5, 1968–1974. (33) Cargile, B. J.; Talley, D. L.; Stephenson, J. L., Jr. Electrophoresis 2004, 25, 936–945. (34) Ernoult, E.; Gamelin, E.; Guette, C. Proteome Sci. 2008, 6, 27. (35) Hubner, N. C.; Ren, S.; Mann, M. Proteomics 2008, 8, 4862–4872. (36) Mulvenna, J.; Hamilton, B.; Nagaraj, S. H.; Smyth, D.; Loukas, A.; Gorman, J. J. Mol. Cell Proteomics 2009, 8, 109–121.


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cIEF, reduced RP-HPLC-MS, intact size exclusion chromatography (SEC)-MS, peptide mapping, bioassay, and N-terminal sequencing (NTS). This work led to the identification of basic charge heterogeneities including noncyclized heavy chain, N-terminal extensions of signal peptide on the heavy chain, and species showing variable C-terminal lysine processing and high-mannose structures. This work also demonstrated that it was feasible to characterize a cIEF profile for a release assay for a monoclonal antibody using material separated under a similar principle of separation. The high level of resolution of the preparative fractionation was a key factor that enabled identification of the species in each peak of the basic charge variants, allowed for correlation of these species to specific peaks in the cIEF profile, and led to an increased understanding of antibody product heterogeneity. EXPERIMENTAL SECTION Materials. Recombinant IgG2 monoclonal antibody was produced in CHO cells and purified according to the method of Shukla et al.37 cIEF reagents were obtained from GE Healthcare (Pharmalyte preparation and IPG strips and buffer), Sigma ((hydroxypropyl)methyl cellulose), Invitrogen (N,N,N′,N′-tetramethylethylenediamine), Convergent Biosciences (pI markers), Bio-Rad (chemical mobilizer), and Teknova (glycerol). Enzymes were purchased from New England Biolabs (PNGase F), Promega (trypsin), and Roche (Glu-C). CE capillaries were purchased from Beckman and chromatography columns were purchased from Poly LC (polyhydroxyethyl aspartamide column), and Varian (Pursuit diphenyl, and PLRP-S columns). N-terminal sequencing reagents were from Applied Biosystems. All reagents used were ACS grade or higher. Capillary Isolectric Focusing. cIEF was performed on a Beckman PA 800 using a neutral-coated (polyacrylamide), 50 µm ID × 30 cm capillary. An ampholyte mixture containing 2.5% (w/ v) Pharmalyte pH 3-10, 0.2% (w/v) (hydroxypropyl)methyl cellulose, 0.3% (v/v) N,N,N′,N′-tetramethylethylenediamine, and pI 6.14 and 8.18 markers was combined with antibody diluted in water to obtain a final antibody concentration of 0.3 mg/mL. Analysis was performed at 25 kV and 20 °C with a 10 min focusing period during which isoforms migrate to their pI in the pH gradient. This was followed by a 25 min mobilization step using the Bio-Rad chemical mobilizer with the UV detector set at 280 nm. OFFGEL Fractionation Using Immobilized pH Gradient. A stock solution was prepared by combining 4.8 mL 50% glycerol, 480 µL of IPG buffer (pH 6-11), and water to produce a total volume of 40 mL. Denaturant and reductant were not included in order to maintain the antibody in native state. The antibody solution to load into the wells was prepared in excess by adding 0.12 mL (9 mg) of purified antibody to 25.9 mL of stock solution and 6.4 mL of water. IPG strip rehydration solution was prepared in excess by combining 1.6 mL of water with 6.4 mL of stock solution. IPG gel strips, pH range of 6.2-7.5, were arranged in every other lane of the two instrument trays, and 24 well frames were snapped in place over them. The standard OFFGEL kit protocol was used for strip rehydration, antibody loading, and (37) Shukla, A. A.; Hubbard, B.; Tressel, T.; Guhan, S.; Low, D. J. Chromatogr., B 2007, 848, 28–39.

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loading of the trays onto the instrument.38 A platform temperature of 30 °C was used. The standard instrument protein focusing method for a 24 well setup was run using a constant current of 50 µA with a max voltage setting of 8000 V and a max power setting of 200 mW. After 40 h of fractionation, the run was stopped and like well numbers from each lane for wells 9 to 20 were pooled, then exchanged into acetate buffer, and concentrated approximately 10-fold prior to analysis. Percent yield for the fractionation was determined by summing the quantity of antibody obtained in each fraction and dividing by the total antibody loaded across the wells. Antibody quantities in each fraction were determined by measuring the absorbance at 280 nm and using the extinction coefficient of the antibody to determine the concentration of the fraction and then multiplying the volume of the fraction by the concentration. Quantities were corrected for the loss due to buffer exchange and concentration. Intact Mass Analysis. Intact samples were deglycosylated using PNGase F to remove N-linked carbohydrates by adding 1 µL of enzyme (500 000 units/mL) per 100 µg of antibody and incubating overnight at 37 °C. Samples were desalted by analytical SEC prior to introduction into an Agilent time-of-flight mass spectrometer using a method we have previously described.39 Briefly, antibody was diluted to 2.0 mg/mL, and then, 4 µg was injected onto a 2.1 mm × 150 mm polyhydroxyethyl aspartamide column. An isocratic elution was performed using 0.1% formic acid. A solution of 2% formic acid in acetonitrile was mixed 1:1 with the HPLC eluent prior to introduction to the electrospray ionization (ESI) source. The Agilent TOF was operated with a spray voltage of 5 kV and a fragmenter voltage of 415 V. Nebulizer pressure was 60 psi and nitrogen source gas flow was 9 L/min. Ion transmission was set by tuning using Agilent autotune, and the mass range was calibrated from 0 to 4000 m/z below 3 ppm accuracy. All data were acquired from 0 to 4000 m/z. Deconvolution was performed using the Agilent MassHunter software. Reduced Mass Analysis. Samples were denatured and reduced in 50 mM DTT, 3 M guanidine, and 50 mM Tris, pH 8, for 30 min at 55 °C prior to analysis. RP-HPLC separation was performed using an injection of 20 µg on a Pursuit diphenyl 2.0 × 150 mm column with 0.1% TFA (w/v) for mobile phase A and 90% acetonitrile with 10% water and 0.1% TFA (w/v) for mobile phase B, on the basis of a method described by Ren et al.40 Briefly, the column was held in 20% mobile phase B for 3 min, followed by a step gradient from 20 to 38% mobile phase B in 2 min, and followed by an elution step of 38 to 44% mobile phase B in 30 min. The column was heated to 75 °C during operation, and the flow rate was 0.3 mL/min. The Agilent TOF instrument was used for mass detection without flow splitting. The Agilent TOF was operated with a spray voltage of 4 kV and a fragmenter voltage of 250 V. Nitrogen flow was 12 L/min. Other settings were determined as for intact mass analysis above. Trypsin and Glu-C Peptide Mapping. Samples were reduced with 10 mM DTT, alkylated with 21 mM iodoacetic acid, and buffer exchanged into 100 mM Tris, pH 7.5, with 0.1 M urea. Samples (38) Agilent 3100 OFFGEL Fractionator Kit Quick Start Guide, 2nd ed.; Agilent Technologies, 2007. (39) Brady, L. J.; Valliere-Douglass, J.; Martinez, T.; Balland, A. J. Am. Soc. Mass Spectrom. 2008, 19, 502–509. (40) Ren, D.; Pipes, G.; Xiao, G.; Kleemann, G. R.; Bondarenko, P. V.; Treuheit, M. J.; Gadgil, H. S. J. Chromatogr., A 2008, 1179, 198–204.

Figure 1. Representative cIEF profile of the antibody of interest for characterization using preparative IEF. The profile shows the basic (22.9 ( 0.8%, pI 7.35 ( 0.02), main (56.9 ( 0.7%, pI 7.21 ( 0.02), and acidic (20.2 ( 0.6%, pI 7.15 ( 0.02) peak groups bracketed by the pI markers (n ) 15).

were then digested with sequence grade modified trypsin or Glu-C using a 1:25 antibody/enzyme ratio for 4 hours at 37 °C and quenched with trifluoroacetic acid. Peptides were separated on a PLRP-S 250 × 2.1 mm column using a 0.2 mL/min flow rate with a mobile phase A of 0.08% TFA (v/v) in water and a mobile phase B of 90% acetonitrile with 10% water and 0.07% TFA (v/v). The column eluate was directed to the ESI source of a ThermoFinnigan LTQ XL ion trap mass spectrometer set to analyze MS and MSn. N-Terminal Sequencing. Samples were subjected to automated Edman degradation 1,2 chemistry on an Applied Biosystems Procise Protein Sequencer. The protein N-terminus was coupled with phenylisothiocyanate (PITC) under basic conditions. The PITC coupled N-terminal amino acid was subsequently cleaved off with neat TFA and converted from an anilinothiozolinone to a phenylthiohydantoin (PTH)-amino acid under acidic conditions. The PTH-amino acids were then separated by RPHPLC on a Brownlee Spheri-5 PTH column with an isopropanol/ acetonitrile gradient in the presence of sodium acetate. The sequence and yield of amino acids were analyzed on model 610A analysis software (Applied Biosystems). PTH-amino acids were identified by their distinct retention times. Potency Assay. Potency was measured by a luciferase reporter gene bioassay. Reference standard antibody and test samples were incubated with the target ligand in the presence of in-house engineered murine BaF cells stably expressing target receptor complex and Stat luciferase gene. Ligand binding to the receptor on the surface of transfected BaF cells results in Stat 5 activation and an increase in luciferase activity. Competitive binding of test antibodies to the ligand inhibits signal transduction and luciferase response in a dose dependent manner. Following incubation, cells were treated with cell lysis buffer containing luciferin, a substrate for luciferase. The reaction of luciferase in lysed cells with luciferin results in luminescence, which was measured using a luminometer. Test sample biological activity was determined by comparing the test sample response to a standard.

RESULTS AND DISCUSSION There are several aspects of cIEF that make it possible to obtain highly resolved, highly reproducible charge isoform profiles for antibodies compared to other methods such as CEX. These include focusing and chemical mobilization under voltage, using a narrow ID capillary (50 µm) and a neutral capillary coating, and using ampholytes to create a pH gradient for focusing. A typical, optimized cIEF profile for the antibody of interest is shown in Figure 1, with two marker pI peaks bracketing the basic isoforms, main peak, and acidic isoforms. The main peak has a pI of 7.2. The main peak accounts for about 60% of the total corrected area, leaving the composition of the remaining 40% in the basic and acidic isoform groups unknown. The goal of this work was to identify the major species present in the acidic and basic group peaks. In this paper, we describe characterization of the basic components of the profile. Efforts to characterize the peaks in the cIEF acidic group are ongoing. Use of the OFFGEL preparative IEF instrument was evaluated to produce pI-based fractions of the antibody for charge isoform characterization. The IPG gel strips used in the instrument contain the pH gradient for separation, making the separation range somewhat flexible based on the strips commercially available. Another advantage of the IPG gel strips is that they have been used for many years in 2-D gel electrophoresis applications and are, thus, well characterized. Antibody fractionation was optimized to maximize peak purity per fraction by varying the IPG gel pH range, the platform temperature, fractionation time, and the load amount per lane. The optimal conditions selected were a pH 6.2-7.5 gel strip, an instrument platform temperature of 30 °C, a fractionation time of 40 h, and a load of 1 mg of antibody per lane in each of 8 lanes (8 mg total loaded). Peptide mapping and potency testing of starting material incubated in the sample matrix at 30 °C for 40 h showed no significant changes from the starting material control, indicating that the fractionation temperature and matrix did not generate artifactual modifications. Analytical Chemistry, Vol. 82, No. 9, May 1, 2010

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Figure 2. cIEF profiles of fractions obtained from an optimized OFFGEL preparative IEF fractionation of the antibody show isolation of the basic, main, and acidic peaks across 12 fractions compared to the starting material. The composition of the isoform present, expressed in percentage, was determined using cIEF peak area and is indicated for fractions 12, 14, and 17.

After fractionation, pools of like wells were analyzed by cIEF to determine purity. Figure 2 shows the resulting cIEF traces from a typical optimized fractionation. A photo of an OFFGEL tray and a schematic of the wells in which the antibody migrates are shown in Figure S-1 (Supporting Information). Reproducible cIEF profiles and yields were obtained for six fractionations run on different days using two different IPG gel lots. The average approximate antibody percent recovery for six fractionations was 51%. Since antibody separated by this method is in equilibrium between the IPG gel strip and the liquid phase and because denaturant and reductant were not included to maintain the antibody in native state, some loss of material to the gel strip was expected. The percent recovery was determined to be sufficient for the present work but could potentially be improved and is an area for future work. The percent yields for the pool of each fraction roughly corresponded to the expected yields based on the cIEF antibody peak area percents for the acidic, main, and basic isoform groups (Table 1). This suggests that all species were similarly wellrecovered. To more clearly show correlation of the fractionated material to the antibody cIEF profile, a spiking experiment was performed by adding 5 or 10% of each fraction into starting material and analyzing the samples by cIEF. This demonstrated correlation of the fractions to the cIEF peaks, as well as the high purity of the fractions (see Figure S-2 (Supporting Information) for representative results for fractions 17 and 18). Characterization of the fractions was then carried out using various analytical techniques. A variety of modifications to the Nand C-termini of the heavy chain were discovered across the basic fractions. The modifications included noncyclized glutamine and 3514


Table 1. Yield Percents for the Preparative IEF Fractions and Peak Groups Compared to the Expected Percentages Based on Peak Area As Measured by cIEF isoform group basic

main acidic

fraction

% of total yielda

20 19 18 17 16 15 14 13 12 11 10 9

1.3 0.3 4.5 8.0 6.2 27.5 24.8 9.1 12.6 2.7 2.4 0.7

sum of yield by isoform group (%)

percent of isoform group in starting material by cIEF (%)

20

24

52

56

27

20

a Antibody quantity in the fraction divided by total antibody recovered from the fractionation.

signal peptide extensions on the N-terminus, as well as variations in lysine processing at the C-terminus. To provide an initial evaluation of differences in heavy or light chain mass between the fractions, reduced reversed-phase liquid chromatography mass analysis was performed. Analysis of reduced samples from fractions 14 (main peak) and 15 to 20 (basic peaks) showed significant differences in heavy chain retention in the UV profile (Figure 3A), which was one of the first indications that antibody containing different heavy chain forms was resolved across the immobilized pH gradient separation. Significant differences in the UV profile were not observed for the light chain.

Figure 3. Reduced mass analysis of fractions 14-20 and the starting material control showed distinct differences in heavy chain (HC) retention by UV (A). The entire heavy chain peak was deconvoluted to produce the mass spectra (B), with mass differences between fractions highlighted by arrows. For fraction 20, several unique masses were observed, as discussed and shown in detail in Figure 6. Masses of the major expected peaks across all the fractions were within 7-14 ppm of expected masses.

Spectra from the entire heavy chain peak for each fraction were summed and deconvoluted to obtain the mass results shown in Figure 3B for fractions 14 through 20 and a starting material control. In these reduced samples, N-linked glycans were left intact on the molecule, conferring some of the observed heterogeneity to the mass data obtained for the heavy chain. The typical level of carbohydrate heterogeneity observed for this antibody is shown in the control sample in Figure 3B (bottom panel), where the major species is antibody with a fucosylated biantennary glycan capped with 0, 1, or 2 galactose residues (abbreviated G0F, G1F, and G2F, respectively). Mass measurement showed that the major antibody species in this sample also contained cyclized N-terminal glutamine and no C-terminal lysine due to cellular processing. Minor species due to terminal galactosylation and high-mannose species were also present. Reduced mass analysis of fraction 14, which contains 89% main peak, showed a similar pattern to the starting material. Masses of the major expected glycan species in each fraction were all within 7-14 ppm of the expected masses. Analysis of fractions 15 and 16 by reduced mass revealed the enrichment of a species having a mass approximately 17 Da above the major G0F heavy chain species. Figure S-3 (Supporting Information) shows a detailed view of the heavy chain deconvolution for fractions 15 and 16 compared to fraction 14 (main peak).

The observed mass shift was suspected to be due to either noncyclized N-terminal glutamine or oxidation. Peptide mapping of these fractions and examination of the MS/MS spectrum associated with the unique UV peak showed y-ion shifts of +17 for the N-terminal peptide compared to the cyclized form, indicating the presence of noncyclized N-terminal glutamine. During antibody cellular processing, the majority of the heavy chain N-terminal glutamine becomes cyclized to pyroglutamic acid,41,42 often leaving a small percentage of antibody noncyclized. The more basic nature of this noncyclized form would cause it to migrate in the basic region of the cIEF profile. Oxidation of a methionine residue in the N-terminal peptide was also observed, but this species was not isolated to a particular fraction. In addition to the N-linked glycan heterogeneity and the species containing noncyclized N-terminal glutamine in fraction 16, an unexpected shift of +89 Da was also observed in the reduced mass results for that fraction (Figure S-3, Supporting Information). Deconvolution of the front shoulder of the heavy chain peak in fraction 17 also showed a shift of +89 Da. Reduced (41) Dick, L. W., Jr.; Kim, C.; Qiu, D.; Cheng, K. C. Biotechnol. Bioeng. 2007, 97, 544–553. (42) Rehder, D. S.; Dillon, T. M.; Pipes, G. D.; Bondarenko, P. V. J. Chromatogr., A 2006, 1102, 164–175.


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Figure 4. Detailed view of the deconvoluted heavy chain spectra for fractions 14, 18, and 19 from the reduced mass analysis shows the enrichment of a +770 Da species in fractions 18 and 19 compared to the main peak in fraction 14.

mass analysis of fractions 18 and 19 showed the presence of another unexpected mass, having a shift of +770 Da, on the heavy chain (Figure 4). Like the +89 Da shift, the +770 Da mass shift had not been previously observed on this antibody and was a surprising observation. Intact mass analysis was run on deglycosylated material from fractions 14 (main peak) and 18 to determine whether the +770 Da modification was present on one or both antibody heavy chains. In addition to unmodified antibody, which had an observed base mass of 144 303.2 Da (17.1 ppm error), a species with a mass shift of +769.8 Da was observed, indicating that the modification was present only on one arm of the antibody (Figure S-4, Supporting Information). Since the carbohydrate heterogeneity present due to N-glycosylation in the Fc domain was removed by treatment with PNGase F, the +769.8 Da species was determined to be unrelated to oligosaccharide heterogeneity. Peptide mapping using Glu-C and trypsin was then utilized as an orthogonal technique to identify the site of the modifications. Peptide mapping revealed that the +89 Da and +770 Da modifications were present on the N-terminal peptide of the heavy chain (H1 peptide). The masses identified were consistent with extensions of the signal peptide sequence prior to the start of the mature heavy chain sequence. The +89 Da shift was correlated to a +1 amino acid extension (alanine), enriched in fractions 16 and 17. (Alanine addition results in +71 Da, and the replacement of the cyclized glutamine by noncyclized glutamine results in +17 Da.) The +770 Da shift was correlated to a +8 amino acid extension, enriched in fractions 18 and 19. (The 8 amino acid extension results in +753 Da, and the replacement of the cyclized glutamine by the noncyclized residue results in +17 Da.) A minor form containing a +7 amino acid extension was also identified in fraction 18. MS/MS results confirmed the expected amino acid sequences for the N-terminal extensions (Figure S-5, Supporting Information). 3516


Additional confirmation of the presence of N-terminal extensions on the heavy chain in fractions 16 and 18 was also obtained using N-terminal sequencing (NTS). Significant levels of the +1 and +8 amino acid heavy chain extensions were detected in fractions 16 and 18, respectively, compared to the main peak fraction. This is consistent with the reduced mass, intact mass, and peptide mapping results. The starting material was also analyzed by N-terminal sequencing, which showed that the sensitivity of the NTS assay was not sufficient to detect signal peptide forms at levels of approximately 8% (by cIEF) in the starting material. Variable C-terminal lysine processing was also observed in several of the basic fractions. Reduced mass analysis of fractions 17 and 18 showed the presence of a heavy chain mass having a +128 Da shift consistent with the presence of C-terminal lysine (Figure S-6, Supporting Information). C-terminal lysine processing in antibodies is a common occurrence believed to be due to basic carboxypeptidase activity,4 though low level populations of antibody often remain that contain intact C-terminal lysine. These variants would also be expected to migrate in the basic region of the cIEF profile. Intact mass analysis of fraction 18 also showed a +128.6 Da shift from the major species, indicating that Cterminal lysine was present on only one arm of the antibody in this fraction (Figure S-4, Supporting Information). Peptide mapping of these fractions also confirmed elevated levels of C-terminal lysine on the C-terminal peptide. Reduced mass analysis of fraction 20, the most basic fraction collected, showed the presence of C-terminal lysine in conjunction with oligomannose glycan variants. These variants showed masses expected for antibody containing C-terminal lysine plus high mannose glycans with 5-9 mannose residues on the heavy chain (Figure 5). Due to material limitations, intact mass was not performed on fraction 20. However, since intact mass analysis of

Figure 5. Heavy chain peak deconvolution results for fraction 20 indicate the enrichment of several oligomannose species containing C-terminal lysine.

fraction 18 showed that the antibody in fraction 18 had only a single C-terminal lysine present on heavy chain, it is probable that fraction 20, containing higher pI variants, is enriched for species in which C-terminal lysine is present on both heavy chains since mannose residues are not expected to contribute to antibody charge heterogeneity. In addition, the reduced mass results for fraction 20 showed higher levels of C-terminal lysine compared to fraction 18, which also suggests that the major species in fraction 20 likely contains C-terminal lysine on both chains. Given that fraction 20 is likely enriched for antibody with two C-terminal lysine residues and oligomannose glycan variants, it is possible that incompletely processed material is present in the cell culture supernatant. These species could correspond to intermediate molecules, stopped at the endoplasmic reticulum (ER) stage during the secretion process and potentially produced during a later stage of the cell culture process when cell viability decreases. Alternately, these two distinct post-translational processes could be linked in the context of expression of this antibody. Gaining further insight into this phenomenon could impact the current understanding of cellular processing and be important to potentially reduce heterogeneity present on therapeutic antibodies produced by CHO cells. Potency analysis was performed on fractions 14-19 using a reporter gene bioassay to determine whether or not the N-terminal extensions or other basic modifications had an impact on ligand binding. The relative potency of fraction 19 (containing 63% of the +8 amino acid extension) was 73 ± 3% compared to the potency of fraction 14 (containing 89% main peak), which was 92 ± 4%. While there was some decrease in the potency of fraction 19 enriched in the +8 amino acid extension compared to main peak, this result shows that the signal peptide extension did not have a major impact on potency. Relative potency of other basic fractions was similar to main peak potency.

To determine the major components of the basic peaks in the cIEF profile, the mass results were correlated to the immobilized pH gradient fraction cIEF peaks. Figure S-7 (Supporting Information) shows how this was done for fractions 16-18. When the same mass was detected in two fractions which also shared a peak by cIEF analysis, the variant having that mass was assigned to the peak shared by the fractions. For example, by mass analysis, the species containing a single C-terminal lysine on the heavy chain was observed in fractions 17 and 18 (Figure S-6, Supporting Information) but not in any other fractions. There was only one cIEF peak that overlapped between fractions 17 and 18 (Figure 2), so the species containing a single C-terminal lysine species was assigned to that overlapping peak, designated B3 (Figure 6). When a unique mass was observed in only a single fraction, it was assigned to the unique cIEF peak observed in that fraction. This strategy allowed for the successful identification of the major forms in each of the basic peaks in the cIEF profile. Figure 6 shows the resulting cIEF profile labeled with the basic variants identified in this work. Theoretical pI values were calculated for each of the variants identified43 and showed good correlation with the experimental values determined by cIEF (Table 2). CONCLUSIONS Charge isoforms of a purified antibody were successfully isolated using the OFFGEL preparative IEF fractionation system and characterized using analytical techniques including cIEF, mass analysis, N-terminal sequencing, and potency analysis. The high (43) Sillero, A.; Ribeiro, J. M. Anal. Biochem. 1989, 179, 319–325. (44) Ying, H.; Liu, H. Immunol. Lett. 2007, 111, 66–68. (45) Lyubarskaya, Y.; Houde, D.; Woodard, J.; Murphy, D.; Mhatre, R. Anal. Biochem. 2006, 348, 24–39. (46) Kao, Y.-H.; Vanderlaan, M. U.S. Patent 7,560,111, 2009. (47) Kotia, R. B.; Raghani, A. R. Anal. Biochem. 2010, 399, 190–195.


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Figure 6. cIEF profile of the antibody showing the major species identified in each basic peak through characterization of the fractions generated using the OFFGEL preparative IEF technique. Table 2. Comparison of Experimental versus Calculated Theoretical pI Values for the Basic Charged Species Identified in the Antibody cIEF Profile peak

experimental pI

main B1 B2 B3 B4 B5

7.21 7.25 7.30 7.36 7.40 7.55

theoretical pI 7.20 7.32 7.32 7.32 7.46 7.46

area percent

(noncyclized Q) (+1 aa) (+K) (+8 aa) (+2K, oligomannose)

56.1 5.2 4.3 6.6 4.4 3.2

purity of these pI-based fractions was significant for this work and allowed for the identification of the major species in each of the five basic peaks observed in the cIEF profile. Basic charge variants identified included signal peptide extensions (+1, 7, and 8 amino acids) on the N-terminus of the heavy chain at levels of approximately 8% by cIEF. Other modifications identified included noncyclized heavy chain (5%), the presence of C-terminal lysine on one arm of the heavy chain (7%), and the presence of C-terminal lysine on both arms of the heavy chain with high-mannose forms (3%). The use of this off-line technique also allowed for the isolation of sufficient quantities of material in the native state to assess the biological potency of the fractions. While the presence of signal peptide extensions has not been widely reported in the literature, extensions have been observed

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for other therapeutic antibodies.44-47 These extensions appear to be the result of improper signal peptide cleavage by the enzyme signal peptidase during cellular processing in the endoplasmic reticulum. Additional work to better understand signal peptide processing, including factors that affect the presence of extensions and the amount present, is ongoing. This work demonstrates the applicability of the OFFGEL immobilized pH gradient fractionation system for the generation of pI-based fractions of antibody charge isoforms which can be used for cIEF profile characterization. ACKNOWLEDGMENT We are grateful to Diana Shpektor for potency analysis and Angie Ziebart for N-terminal sequencing analysis. We thank Rohini Deshpande and the colleagues on her team for scientific discussions and Michael Treuheit for critical reading of the manuscript. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review October 23, 2009. Accepted March 21, 2010. AC902408R

Characterization of Antibody Charge Heterogeneity Resolved by

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