Kinetics of Chemical Degradation in Monoclonal Antibodies

Anal. Chem. 2010, 82, 3198–3206

Kinetics of Chemical Degradation in Monoclonal Antibodies: Relationship between Rates at the Molecular and Peptide Levels Roxana Ionescu* and Josef Vlasak Merck Research Laboratories, Merck & Co. Inc., West Point, Pennsylvania 19486 This article describes a method to analyze the kinetics of monoclonal antibody degradation and to determine the quantitative relationship between the degradation rates observed at the molecular and peptide levels. The proposed model can be applied to any degradation pathway that can be well approximated by a first order reaction; if several pathways exist, the model assumes that they are independent. Three examples are presented to illustrate the benefits of this approach. For each case, the calculated fractions of species were compared to one or more data sets to demonstrate the good agreement between experimental results and model prediction. This method can serve as a valuable tool in interpreting chromatograms of degraded samples and predicting the population of species present at the molecular level when only data from degradation observed at the peptide level is available. This method further demonstrates how deviations from predictions of simple models can be used to unravel additional, unforeseen reactions. With 25 products on the market and hundreds more in clinical trials, monoclonal antibodies are the fastest growing class of human pharmaceuticals, reaching worldwide sales of $20 billion in 2007.1 The outstanding performance of monoclonal antibodies in the drug market is supported, in part, by a substantial scientific effort to understand the structural heterogeneity and stability of this class of complex molecules. Characterizing the structural heterogeneity of the ensemble of molecules that represent “the drug” introduces a challenge in monoclonal antibody development. There are many chemical and physical modifications that the protein can undergo during manufacture and/or storage: asparagine deamidation,2,3 isoasparatate or succinimide formation,4,5 tryptophan6 or methionine7,8 oxidation, cysteinylation,9 glycation,10 pyroglutamate formation,11,12 * To whom correspondence should be addressed. Phone: 215-652-7309. Fax: 215-993-3348. E-mail: [email protected]. (1) Beck, A.; Wagner-Rousset, E.; Bussat, M. C.; Lokteff, M.; Klinguer-Hamour, C.; Haeuw, J. F.; Goetsch, L.; Wurch, T.; Van Dorsselaer, A.; Corvaia, N. Curr. Pharm. Biotechnol. 2008, 9, 482–501. (2) 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 2001, 752, 233–245. (3) Vlasak, J.; Bussat, M. C.; Wang, S.; Wagner-Rousset, E.; Schaefer, M.; Klinguer-Hamour, C.; Kirchmeier, M.; Corvaia, N.; Ionescu, R.; Beck, A. Anal. Biochem. 2009, 392, 145–154. (4) Aswad, D. W.; Paranandi, M. V.; Schurter, B. T. J. Pharm. Biomed. Anal. 2000, 21, 1129–1136.

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disulfide shuffling,13 and hydrolysis.14,15 More details on these structural variants can be found in a review16 describing sources of heterogeneity of monoclonal antibodies. Because chemical and physical modifications may impact the function and/or safety of the biological product, there is a strong need to understand and characterize the degradation mechanisms of monoclonal antibodies. During production in cell culture, the monoclonal antibody protein is exposed for extended durations (e.g., several weeks) to relatively high temperatures (e.g., ∼37 °C), to the complex chemical composition of cell culture media, and to enzymes from the host cells. Because all of these conditions are known to accelerate rates of chemical degradation, it is expected that some heterogeneity is induced during manufacture of monoclonal antibodies. Strategies for improving the quality of recombinant proteins expressed in mammalian cells have been reviewed recently.17 The chemical heterogeneity induced in a monoclonal antibody in cell culture may be reduced to low levels during purification or deemed tolerable if it does not affect product efficacy or safety. Chemical changes of monoclonal antibodies may also accumulate during time in storage, but in the absence of host enzymes and in a solvent with well-defined composition, the heterogeneity induced in this case is limited to fewer degradation pathways than during production. The heterogeneity of monoclonal antibodies can be resolved using different techniques, and various species can be separated (5) Xiao, G.; Bondarenko, P. V.; Jacob, J.; Chu, G. C.; Chelius, D. Anal. Chem. 2007, 79, 2714–2721. (6) Wei, Z.; Feng, J.; Lin, H. Y.; Mullapudi, S.; Bishop, E.; Tous, G. I.; CasasFinet, J.; Hakki, F.; Strouse, R.; Schenerman, M. A. Anal. Chem. 2007, 79, 2797–2805. (7) Lam, X. M.; Yang, J. Y.; Cleland, J. L. J. Pharm. Sci. 1997, 86, 1250–1255. (8) Liu, D.; Ren, D.; Huang, H.; Dankberg, J.; Rosenfeld, R.; Cocco, M. J.; Li, L.; Brems, D. N.; Remmele, R. L. Biochemistry 2008, 47, 5088–5100. (9) Banks, D. D.; Gadgil, H. S.; Pipes, G. D.; Bondarenko, P. V.; Hobbs, V.; Scavezze, J. L.; Kim, J.; Jiang, X. R.; Mukku, V.; Dillon, T. M. J. Pharm. Sci. 2008, 97, 775–790. (10) Brady, L. J.; Martinez, T.; Balland, A. Anal. Chem. 2007, 79, 9403–9413. (11) Yu, L.; Vizel, A.; Huff, M. B.; Young, M.; Remmele, R. L.; He, B. J. Pharm. Biomed. Anal. 2006, 42, 455–463. (12) Dick, L. W.; Kim, C.; Qiu, D. F.; Cheng, K. C. Biotechnol. Bioeng. 2007, 97, 544–553. (13) Liu, Y. D.; Chen, X.; Enk, J. Z.; Plant, M.; Dillon, T. M.; Flynn, G. C. J. Biol. Chem. 2008, 283, 29266–29272. (14) Davagnino, J.; Wong, C.; Shelton, L.; Mankarious, S. J. Immunol. Methods 1995, 185, 177–180. (15) Harris, R. J. J. Chromatogr., A 1995, 705, 129–134. (16) Liu, H.; Gaza-Bulseco, G.; Faldu, D.; Chumsae, C.; Sun, J. J. Pharm. Sci. 2008, 97, 2426–2447. (17) Jenkins, N.; Meleady, P.; Tyther, R.; Murphy, L. Biotechnol. Appl. Biochem. 2009, 53, 73–83. 10.1021/ac902752e  2010 American Chemical Society Published on Web 03/19/2010

based on size, charge, conformation, hydrophobicity, and any combination of these properties.18,19 One approach is to monitor protein heterogeneity on the whole antibody molecule, which will be further referred to as the “molecular level”. In an alternative approach, the protein is digested using proteases prior to analysis and the degradation is monitored on the peptide fragments; this will be further referred to as the “peptide level”. Residues that can undergo chemical modification are called “hot spots”, and the number of potential chemical degradation pathways for a given therapeutic protein is determined by the number of distinct hot spots it contains. IgG monoclonal antibodies are composed of two identical heavy chains and two identical light chains covalently linked by disulfide bonds. If a particular IgG residue is prone to chemical modification, there will be two copies of this hot spot in each antibody molecule, both associated with the same degradation pathway. Subsequently, two species will be observed at the peptide level as a result of chemical degradation: the “native” form and the “degraded” form containing the hot spot. However, a single hot spot will result in three species at the molecular level: the “native” protein, the protein with one hot spot modified, and the protein with both hot spot sites modified. Thus, for monoclonal antibodies, the kinetics observed at the peptide level are different from the kinetics observed at the molecular level. The model presented in this paper describes the heterogeneity of monoclonal antibodies expected to accrue over time and provides the quantitative relationship between the kinetics at the molecular and peptide level. Several examples are presented with data from evaluation of monoclonal antibodies, though the model may be applied to any protein with pairs of identical and independent hot spots. To our knowledge, no other published work provides the exact solutions for the time-dependence of the species for such systems. Therefore, the information provided in this article can be used as a tool to analyze data generated by scientists working on the characterization and formulation of monoclonal antibodies. Understanding the kinetics of chemical reactions that are prevalent in a particular monoclonal antibody is critical for defining the shelf life of the drug product. The model also demonstrates how the kinetic approach can help to better understand and characterize the degradation pathways of monoclonal antibodies. Finally, this tool is useful to quantitatively translate observations made at the peptide level to expected heterogeneity at the molecular level.

over time based on the rates of interconversion kji, where kji is the rate of degradation of the hot spot observed at the peptide level. The time-dependence of the population of all species can be described by a system of linear differential equations of the form20 d[Si] ) dt

(∑ )

kji[Sj] - [Si]

j*i

∑k

ij

(1)

j*i

where Si and Sj represent molar fractions of any two species in the ensemble that contain distinct patterns of degraded hot spots and kji is the rate describing the conversion of species Sj into species Si as a result of the degradation of a specific hot spot: kji

(2)

SjfSi

For any pair Sj and Si with a nonzero kji rate, the reverse rate (conversion of Si into Sj) is considered zero. The system of linear differential equations was solved following classical procedures.21 The solutions [Si](t) are expressed as a sum of exponentials of the form: [Si](t) )

∑R

ij

exp(-λjt)

(3)

j

METHODS The kinetic model described herein is valid for reactions that can be well described as “first order” and is based on the assumption that degradation at one site does not affect the degradation rate at another site. The hot spots can be located anywhere in the molecule, in the light chain or heavy chain, in the variable or constant regions, as long as the degradation reactions can be considered independent. A protein with all of its hot spots in the nondegraded form will be called “native” or “intact”. The native protein and the products of the degradation reactions at the molecular level form an ensemble that evolves

The rate constants λj are the rates observed at the molecular level and represent linear combinations of the rate constants kji defined at the peptide level. For a system comprising “n” distinct species at the molecular level, there are “n” values for λj (one solution is always zero, corresponding to the equilibrium value). The coefficients Rij are obtained from eqs 1 and 3 using also the initial (t ) 0) and equilibrium (t ) ∞) conditions. Application of this model is illustrated through the analysis of the three cases. Case I: One Hot Spot with One Degradation Product. This model may be applied to monoclonal antibodies with one hot spot. At the peptide level, the native sequence, “A”, undergoes a chemical modification with the rate constant “k”, resulting in species “X”. Chemical degradation of this particular residue results in the presence of the following species (at the molecular level) at any given time: AA, AX, XA, and XX. In this notation, “AA” represents intact monoclonal antibody molecules, “AX” and “XA” represent antibody molecules that have one intact chain and one chemically degraded chain, and “XX” represents antibody molecules with both chains chemically degraded at the given hot spot. The kinetic scheme for the chemical degradation of a monoclonal antibody with one hot spot is illustrated in Scheme 1; the native chains are shown in dark color and the modified/degraded chains are shown in light color. AX and XA cannot be distinguished chromatographically, as XA is the mirror image of AX along a symmetry axis defined by the hinge region and the CH3/CH3 interface. These species illustrate that any molecule can degrade either in the “left arm” or the “right arm” (left or right can be assigned to each molecule

(18) Vlasak, J.; Ionescu, R. M. Curr. Pharm. Biotechnol. 2008, 9, 468–481. (19) Valliere-Douglass, J.; Wallace, A.; Balland, A. J. Chromatogr., A 2008, 1214, 81–89.

(20) Bilsel, O.; Zitzewitz, J. A.; Bowers, K. E.; Matthews, C. R. Biochemistry 1999, 38, 1018–1029. (21) Ikai, A.; Tanford, C. J. Mol. Biol. 1973, 73, 145–163.


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Scheme 1. Kinetic Scheme for Monoclonal Antibodies with One Hot Spot and One Degradation Producta

a

The chains in light color contain the hot spot degraded.

by an imaginary snapshot at t ) 0). The fact that XA and AX are a priori experimentally indistinguishable is a direct consequence of the fact that “left” and “right” are defined based on an imaginary snapshot at t ) 0 and do not represent an intrinsic asymmetry of the molecule. Therefore, the following species may be experimentally resolved: native (AA), monodegraded (the sum of the species XA and AX), and double-degraded (XX). The molecular rate constants for this system, the time-dependence of each species, and a spreadsheet containing the relevant equations are provided in the Supporting Information. Case II: Two Hot Spots with One Degradation Product Each. In this case, the monoclonal antibody molecule has two hot spots, “A” and “B”, and each of these hot spots can have only one degradation product. At the peptide level, this case can be described as follows: “A” can undergo a chemical modification with the rate constant “ka”, resulting in species “X”, and “B” can undergo a chemical modification with the rate constant “kb”, resulting in species “Y”. The type of chemical reaction that occurs at site “A” may be different from the chemical reaction that occurs at site “B”. At the molecular level, the complexity of the system increases significantly compared with case I. The cascade of kinetic events for two degradation sites with one degradation product each is described in Scheme 2. The native protein is

designated “AABB”, and the positions in this four-letter scheme are assigned based on whether the site is located on the left or right arm; for instance, “XABB” is a molecule with site A degraded in the left arm, “XXBY” is a molecule with site A degraded in both chains and site B degraded in the right arm only, and so on. According to this notation, Scheme 2 results in the presence of 16 species, though only 10 are distinct species that may be distinguished experimentally. The species formed in this case include the following: “native” (AABB), “monoA” (AXBB and XABB), “monoB” (AAYB and AABY), “doubleA” (XXBB), “doubleB” (AAYY), “double_Mix1” (AXBY + XAYB, changes occurring in either the left or right half of the molecule), “double_Mix2” (XABY + AXYB, changes occurring in both left and right halves of the molecule), “tripleA” (AXYY + XAYY), “tripleB” (XXBX + XXXB), and “allD” (XXXX). In principle, the species with degradation at both A and B sites present in the same arm (double_Mix1) may interact differently with columns and be separated from the species that have degradation at A and B sites in separate arms (XABY and AXYB). If the separation method cannot resolve double_Mix1 from double_Mix2, then only nine species will be detected and “double_mix” will represent the sum of double_Mix1 and double_Mix2. Practically, the separation of all species is a very challenging analytical task that depends not only on the skills of the analyst for the selection of proper separation conditions but also on the type of degradation reactions and on the location of the A and B sites. The molecular rate constants for this system, the time-dependence of each species, and a spreadsheet containing the equations are provided in the Supporting Information. Case III: One Hot Spot with Two Degradation Products. In this case, the monoclonal antibody has one hot spot, “A”, that can undergo chemical degradation to form either species “X” or “Y”. Such a situation may be encountered when deamidation of an asparagine residue (Asn) results in either aspartate (Asp) or isoaspartate (IsoAsp), or when a methionine residue is oxidized to S- or R-sulfoxide forms. The rate of formation of species X is

Scheme 2. Kinetic Scheme for Monoclonal Antibodies with Two Hot Spots and One Degradation Product Each

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Scheme 3. Kinetic Scheme for Monoclonal Antibodies Having One Hot Spot and Two Degradation Products with (Right Panel) or without (Left Panel) Interconversion between Species

“kx” and the rate of formation of species Y is “ky”. The corresponding cascade of kinetic events for one degradation site with two degradation products is described in the left panel of Scheme 3. In some cases, an additional reaction may take place due to interconversion of species Y to species X. The corresponding cascade of kinetic events for one degradation site with two degradation products and interconversion among products is described in the right panel of Scheme 3. If species X and Y can be resolved experimentally, the degradation of a monoclonal antibody with one hot spot and two degradation products will result in six distinct species: “native”, “monoX” (AX and XA), “monoY” (AY and YA), “doubleX” (XX), “doubleY” (YY), and “double_Mix” (XY and YX). The rate constants at the molecular level for Scheme 3 (with/without interconversion) and the timedependence of each species were determined using the same method as that described for the previous two cases. RESULTS AND DISCUSSION For each of the cases presented below, the degradation reactions will be defined at the peptide level by indicating the number of hot spots, the products of the chemical modifications, and the corresponding kinetic rates and at the molecular level by describing the number of species and their time dependence. Case I: One Hot Spot with One Degradation Product. In this case, there is one dominant degradation pathway for the monoclonal antibody: at the peptide level, the hot spot “A” undergoes a chemical modification with the rate “k”, resulting in the product “X”. At the molecular level, there can be three species present in solution: “native” (unmodified protein, designated AA), “monoD” (protein with one site modified, designated AX and XA), and “doubleD” (the protein with both sites modified, designated XX). Because of the complexity of monoclonal antibody molecules, one may argue that a single hot spot is very unlikely to be representative of this type of protein because there are typically many sites that can undergo chemical modifications.16 Although this is often the case, there are many examples in which the “one hot spot with one degradation product” model may be a good approximation to describe the degradation of monoclonal antibodies. Such situations can be encountered either when one degrada-

tion rate is much faster than others or when the analytical method used to monitor degradation is sensitive to only one chemical modification. The first example illustrating a case when the degradation rate for one hot spot is much faster than others is a decysteinylation reaction under mild reducing conditions. It was recently reported that an IgG1 monoclonal antibody was partially cysteinylated on an unpaired cysteine residue in the variable regions.9 The decysteinylation reaction was initiated by spiking free cysteine into a solution containing cysteinylated molecules at pH 7.5 and then quenched at different time points by lowering the pH. The fraction of “native” (protein with both sites cysteinylated), “monoD” (protein with only one site cysteinylated), and “doubleD” (protein with no cysteinylation) was monitored by liquid chromatography-mass spectrometry (LC-MS). The evolution in time of the population of these species is shown in Figure 1. The experimentally determined fraction of each species (symbols in Figure 1) were recalculated by subtracting the background (less than 10%) and renormalizing the data presented in the original report.9 The lines

Figure 1. Time dependence of the population of different species during the decysteinylation reaction. Symbols represent experimental values reported in the literature9 normalized after background correction. Lines represent predicted values using the “one hot spot with one degradation product” model: native (protein with both sites cysteinylated) in b and solid line, monoD (protein with only one site cysteinylated) in O and dotted line, and doubleD (protein with no cysteinylation) in 1 and dashed line. Analytical Chemistry, Vol. 82, No. 8, April 15, 2010

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in Figure 1 were obtained from our model by considering the reaction rate at the peptide level that corresponds to the modification of the cysteinylated site to the uncysteinylated product. As an example of the analytical procedure, in the Supporting Information is shown the system of differential equations (eqs 1 and 2 for the particular case of Scheme 1), and the corresponding solution (eq 3 for the particular case of Scheme 1). There is a very good agreement between the experimental and theoretical values. The time dependence of all three species in solution could be fitted simultaneously with only one rate constant (k ) 0.078 min-1) using the equations shown in the Supporting Information (the fraction of native at t ) 0 is 95%) and performing global analysis on the entire data set with the SigmaPlot program. The results in Figure 1 illustrate the expected variation in time of the fraction of species present in solution when there is one hot spot in a monoclonal antibody that can undergo a chemical reaction. The population of the native protein (in the case of the decysteinylation reaction, the native is the “undesired” form of fully cysteinylated molecule) decreases steadily in time. The population of the monoD species (protein with only one site decysteinylated) increases in the first part of the process, reaches a maximum of 50%, and then decreases. The population of the doubleD species (protein with both sites decysteinylated) presents a lag phase in its formation and becomes the dominant species toward the end of the process. The lag phase in the formation of doubleD demonstrates that the probability of formation of species with modifications in both sites is very low unless a significant amount of monoD has accumulated. The equations describing this model (see the Supporting Information) explicitly indicate that, at the molecular level, only the native protein presents a simple exponential decay with twice the rate observed at the peptide level. The time dependence of monoD and doubleD is described by a complex combination of exponential functions. In early stages of degradation (i.e., conditions under which the approximation exp(-Rt) ∼ (1 - Rt) is valid), the decrease of the fraction of intact molecule as a function of time can be approximated with a linear function having the slope “-2k”, the increase of monoD versus time can be described by a line with the slope “2k”, and the fraction of doubleD is zero. This relationship may be a useful tool to predict the shelf life of a monoclonal antibody when the rate of degradation is measured at the peptide level; at the molecular level, the degradation rate is twice that observed at the peptide level. For some proteins, the shelf life is defined as the time when 90% of the protein is in the native form.22 For a monoclonal antibody, with 90% of the molecules in the native form, this corresponds to the case where only 5% of the hot spot population is in the degraded form. This model can be a very useful tool because one can predict the population of species present at the molecular level when only data from degradation observed at the peptide level is available. If degradation is monitored using peptide maps, the information at the molecular level is lost after trypsin digestion; however, it can be “reconstructed” using the equations provided by our model. Table 1 presents a few examples of the fraction of native, monoD, and doubleD species predicted as a function of the fraction of the intact hot spot. For instance, if peptide analysis finds about 30% (22) Roberts, C. J.; Darrington, R. T.; Whitley, M. B. J. Pharm. Sci. 2003, 92, 1095–1111.

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Table 1. Fraction of Native, monoD, and doubleD As a Function of Intact Hot Spot intact hot spot

native

monoD

double D

0.85 0.70 0.49 0.31 0.23

0.73 0.49 0.24 0.10 0.05

0.25 0.42 0.50 0.43 0.35

0.02 0.09 0.26 0.47 0.60

of the hot spot to be degraded (i.e., 70% of the hot spot is in the intact form), then one would expect that a chromatographic technique on the whole protein can resolve three peaks with the relative amplitudes corresponding to 49% native, 42% of the protein with degradation in only one chain, and only 9% of the protein with degradation in both chains. The percentages shown in Table 1 for the case “one hot spot with one degradation product” can be retrieved using a different approach.23,24 If the fraction of intact hot spot is “P” and the fraction of degraded hot spot is “1 - P”, then the probability of each species is as follows: P2 for native, 2P(1 - P) for monoD, and (1 - P)2 for doubleD (see the Supporting Information for details on the equations). The example provided by Banks et al.9 on decysteinylation is an outstanding case where the reaction goes to completion (∼100% doubleD) in a very short time (∼1 h) under mild solvent conditions where other chemical modifications are not expected to occur in the monoclonal antibody. The model describing “one hot spot with one degradation product” can be applied more often in the early stages of a degradation process when, under certain stress conditions, one degradation pathway is dominant. The kinetic model can predict the fraction of doubleD as a function of the fraction of monoD detected; this is a very powerful tool when the identity of the species observed at the molecular level during forced degradation is not known. For instance, if variants of a monoclonal antibody are separated by chromatography after some type of stress, one can determine whether two peaks are due to the same hot spot based solely on amplitude considerations. Figure 2 illustrates the good agreement between the data in the literature and the predicted fraction of doubleD as a function of monoD for different degradation pathways. One example (circles, Figure 2) describes the deamidation reaction at Asn30 in the complementarity determining region 1 (CDR1) of the Herceptin light chain;2 another example (triangles, Figure 2) describes the accumulation of succinimide from Asp30 in an IgG2 molecule stored under mildly acidic conditions.24 In both cases, the monoD and doubleD were resolved from the native protein by cation-exchange chromatography (CEX). In another report,19 single and double degradation events due to Asp isomerization or succinimide formation were resolved by hydrophobic interaction chromatography. The agreement between predicted and observed values is also demonstrated for clipping in the hinge region,25,26 another degradation mechanism to which monoclonal antibodies are susceptible. Fab, Fab-Fc, and Fc (23) Shen, J. F.; Kwong, M. Y.; Keck, R. G.; Harris, R. J. Techniques in Protein Chemistry VII; Academic Press, Inc.: San Diego, CA, 1996. (24) Chu, G. C.; Chelius, D.; Xiao, G.; Khor, H. K.; Coulibaly, S.; Bondarenko, P. V. Pharm. Res. 2007, 24, 1145–1156. (25) Cordoba, A. J.; Shyong, B. J.; Breen, D.; Harris, R. J. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 818, 115–121.

Figure 2. Dependence of doubleD species on the fraction of monoD. Line: predicted values based on the “one hot spot with one degradation product” model. Symbols: experimental values reported in literature for deamidation2 (b), succinimide formation24 (1), or fragmentation in the hinge region (g, data from this paper).

fragments form as result of fragmentation in the hinge and the reaction can be monitored by size-exclusion chromatography. The data (g in Figure 2) were obtained for an IgG1 molecule stored at 45 °C; details on the experimental method are provided in SI. Case II: Two Hot Spots with One Degradation Product Each. In this case, there are two major degradation pathways at the peptide level: the hot spot “A” undergoes a chemical modification with the rate “ka”, resulting in the product “X”, and the hot spot “B” undergoes a chemical modification with the rate “kb”, resulting in the product “Y”. At the molecular level, there can be ten distinct species present in solution (for the structure of these species, see description in the methods section and SI). The number of species detected in solution at a given time depends on the sensitivity of the analytical methods to resolve different variants and on the progress of the degradation reaction. As is illustrated below, the species with limited modifications are populated at the beginning of the degradation process. However, species containing three or more modified hot spots become significantly populated only later in the process, which is typically not reached in routine stability protocols. In theory, this model can be applied in all cases for which two degradation rates are independent of each other and significantly larger than other degradation rates that may occur in a monoclonal antibody. In practice, because of the analytical challenges associated with resolving numerous (∼10) distinct species, there are few examples containing a detailed description of the time dependence of population of species in solution. Among these, there is an outstanding report in the literature presenting the degradation of a murine monoclonal antibody27 that very effectively illustrates the “two degradation hot spots with one degradation product each” model. Two hot spots, Asp161 in the light chain and Asp141 in the heavy chain, were found to increase, due to deamidation, the charge heterogeneity of a murine monoclonal antibody.27 The protein was stored in a buffer at pH 9.5 and 25 °C for up to 120 h, aliquots were removed, and the reaction was quenched by dropping the pH to 5.0. Nine different species were resolved by (26) Cohen, S. L.; Price, C.; Vlasak, J. J. Am. Chem. Soc. 2007, 129, 6976– 6977. (27) Perkins, M.; Theiler, R.; Lunte, S.; Jeschke, M. Pharm. Res. 2000, 17, 1110– 1117.

CEX; each fraction was collected, and the identity of each species was resolved by peptide mapping. The CEX chromatograms present three main peaks that correspond to species with (at the molecular level) zero, one, or two deamidations at Asn161 in the light chain (these species were indexed with Roman numerals I, II, and III, respectively). Each of these main peaks contain three peaks, each corresponding to species having zero, one, or two deamidations at Asn141 in the heavy chain (these species were indexed with Arabic numerals 1, 2, and 3, respectively). The correspondence between the nomenclature of species presented in Scheme 2 and the index in the cited report is the following: I-1 is native, I-2 is monoA, I-3 doubleA, II-1 is monoB, II-2 is double mix, II-3 is tripleB, III-1 is doubleB, III-2 is tripleA, and III-3 is allD. We integrated the chromatograms reported by Perkins et al.27 for different times during high-pH, high-temperature stress, and the time dependence of the population of each species is shown in Figure 3A-C. The graphs were grouped based on the number of hot spots degraded (zero or one in panel A, two in panel B, and three or four in panel C) and not on the degradation pathway. Lines on the same graphs show the prediction of the “two hot spots with one degradation product each” model. The results indicate that the entire data set can be very well described simultaneously with only two parameters: the rates ka (0.230 days-1) and kb (0.089 days-1). Details of the equations used for fitting are provided in the Supporting Information. In the case of the murine antibody with two major degradation pathways due to Asp deamidation,27 the ratio of the rates of degradation at the peptide level of the two hot spots is about 2.6. In this particular case, the decrease of native state is concurrent with an increase (up to ∼30%) in monoA, the quickly degrading species with one modified hot spot, and only a small increase (up to ∼10%) in monoB, the slowly degrading species with one modified hot spot. When only 10% of the native molecule remains, the doubleA, double mix, and tripleB species become significantly populated (∼20% each). All other species are present at low levels (less than 10%) during the progress of the reaction. It is interesting to note that the chromatographic method did not separate the double_Mix1 species (molecules with both hot spots monodegraded and changes occurring in either left or right half of the molecule) from the double_Mix2 species (molecules with both hot spots monodegraded and changes occurring in both left and right half of the molecule); they all eluted under one peak that we will refer to as “double mix”. The impact of the ratio ka/kb on the population of different species was calculated for two distinct situations: (1) ka and kb are close in value (ka/kb ) 1.5) and (2) ka is larger than kb (ka/ kb ) 5). The results are presented in Figure S-1 in the Supporting Information. When ka is much larger than kb, the corresponding degradation pathway becomes dominant and the loss of native protein can be well described with the “one hot spot with one degradation product” model. The “two hot spots with one degradation product each” model is based on the assumption that degradation of the hot spots A and B are independent (i.e., the degradation rates ka and kb are constant throughout the entire degradation process). In the case of the murine antibody deamidation reported in the literature,27 this proved to be a valid assumption. Although, in principle, degradation at one site may induce conformational changes to Analytical Chemistry, Vol. 82, No. 8, April 15, 2010

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Figure 3. Time dependence of the charge variants produced by deamidation of two independent hot spots in a murine monoclonal antibody. Symbols represent fractions calculated from the chromatograms published in the literature.27 Lines represent predictions of the “two hot spots with one degradation product each” model.

alter the degradation rate at the other site, systematic studies on the time dependence of different species for such cases were not, to our knowledge, reported in the literature. There is a report where degradation at one site was found to be correlated with degradation at an adjacent site in the case of photoinduced oxidation of methionine residues in the Fc fragment of monoclonal antibodies,28 but the mechanism of the degradation reaction is not understood. The deamidation reaction can result in two degradation products, Asp or IsoAsp, and for unstructured peptides, the ratio (28) Liu, H.; Gaza-Bulseco, G.; Zhou, L. J. Am. Soc. Mass Spectrom. 2009, 20, 525–528.

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of these species is typically 1:3.29 In proteins, because of the constraints imposed by the protein structure, the ratio of aspartate/isoaspartate can vary significantly and there can be instances where only one of these species is formed.30 It is unclear why, for the case presented above, the deamidation of the hot spots results in only one degradation product. It may be possible that the protein structure favors formation of only one species (Asp or IsoAsp) or that the chromatographic method cannot resolve species that contain Asp or IsoAsp at the degraded hot spot. If the deamidation observed in the murine monoclonal would have produced two degradation products for each hot spot, the complexity of the reaction would have increased tremendously. A more simple case with only one hot spot and two degradation products will be presented below. Case III: One Hot Spot with Two Degradation Products Each. This section illustrates the application of kinetic modeling in the particular case in which deamidation at one hot spot can result in several degradation products. There are many examples in the literature of deamidation of Asn residues in monoclonal antibodies that may result in the formation of Asp, IsoAsp, and even accumulation of succinimide.3,24 The nature of reaction products depends on the protein structure or on solvent conditions. In the constant regions of monoclonal antibodies located in the Fc fragment, two hot spots prone to deamidation present only one deamidation product, most likely due to structural constraints: Asn382 degrades into IsoAsp and Asn387 degrades into Asp.30 Similar observations were made for a monoclonal antibody that contains the hot spot in the variable regions: only the Asp-containing form was detected upon deamidation.2 Succinimide product was found to accumulate mainly under mildly acidic conditions,24 but detection of this deamidation intermediate at the peptide level is challenging due to rapid breakdown of succinimide to Asp or IsoAsp in unstructured peptides.5 The rate of deamidation depends on solvent conditions and protein structure.31 In monoclonal antibodies and other proteins as well, Asn residues in more flexible and solvent exposed regions (like the CDRs) tend to have higher deamidation rates.32 Deamidation in the CDRs may3 or may not2 change binding affinity of the antibody. It is also possible that deamidation may change the immunogenicity of the protein, as in the case of Celiac disease.33 The example presented here describes Asn deamidation of a hot spot in CDR1 of an IgG1 molecule that results in the formation of two products: Asp and IsoAsp (no succinimide detected). Regarding the nomenclature defined in the Methods section, the molecules having only one hot spot deamidated will be referred to as “monoX” if the degraded hot spot contains IsoAsp and “monoY” if the degraded hot spot contains Asp. For this particular IgG1 molecule, monoX and monoY species can be well separated by CEX, present conformational changes revealed by near-UV CD (29) Patel, K.; Borchardt, R. T. Pharm. Res. 1990, 7, 703–711. (30) Sinha, S.; Zhang, L.; Duan, S.; Williams, T. D.; Vlasak, J.; Ionescu, R.; Topp, E. M. Protein Sci. 2009, 18, 1573–1584. (31) Robinson, N. E.; Robinson, A. B. Molecular Clocks: Deamidation of Asparaginyl and Glutaminyl Residues in Peptides and Proteins; Althouse Press: Cave Junction, OR, 2004. (32) Robinson, N. E.; Robinson, A. B. Proc. Natl. Acad. Sci. U.S.A 2001, 98, 4367–4372. (33) Henderson, K. N.; Tye-Din, J. A.; Reid, H. H.; Chen, Z.; Borg, N. A.; Beissbarth, T.; Tatham, A.; Mannering, S. I.; Purcell, A. W.; Dudek, N. L.; van Heel, D. A.; McCluskey, J.; Rossjohn, J.; Anderson, R. P. Immunity 2007, 27, 23–34.

Figure 4. Time dependence of fraction of molecules with deamidated hot spot in only one arm of the monoclonal antibody, resulting in either IsoAsp (3) or Asp (b) at pH 6.5 or pH 8.0. The lines represent results of data analysis using Scheme 3 with (solid lines) or without (dashed lines) interconversion of IsoAsp into Asp.

and different susceptibility to proteases, and have significantly different thermal stabilities.3 Figure 4 shows the formation of monoX (3) and monoY (b) species when the sample was stored under accelerated conditions in phosphate buffer at pH 6.5 or pH 8.0. As expected, after 1 month of storage at 37 °C, the total fraction of deamidated species is much higher at pH 8.0 than at pH 6.5, simply because the deamidation reaction is strongly accelerated by increasing pH.34 Despite the significant variation in the deamidation rate, both solvent conditions present the same interesting trend: there is an initial, more rapid accumulation of the species containing IsoAsp (3 in Figure 4) compared to formation of Asp (b in Figure 4), but at later time points, there is a “crossover” point and the molecules containing Asp become predominant. Analysis of the data at pH 6.5 using the left panel of the Scheme 3 “one hot spot, two degradation products without interconversion between species” model provides the following rates: kx ) 0.0017 days-1, ky ) 0.0021 days-1, and the fit it shown in Figure 4 as dashed lines. Because a significant fraction of native, intact protein was still present at the end of the process (∼75%), the population of the species containing both hot spots deamidated (doubleX, doubleY, or doubleMix) was considered negligible at pH 6.5. The fit shown with dashed lines provides a rough description of the formation of deamidated products but does not follow the subtle curvatures of the plots and does not predict the crossover point. The fit improves significantly if the kinetic scheme is modified to include a rate of interconversion of the IsoAsp species into Asp, as shown in the right panel of Scheme 3 the “one hot spot, two degradation products with interconversion between species” (34) Peters, B.; Trout, B. L. Biochemistry 2006, 45, 5384–5392.

model. The results of the fit are shown as a solid line in Figure 4, and the rates that were obtained in this case were kx ) 0.0014 days-1, ky ) 0.0025 days-1, and k ) 0.0134 days-1. The magnitude of the kx and ky rates was not significantly affected by considering the interconversion rate, but the crossover point could be well predicted using this model. As was presented elsewhere,3 this model can be successfully used to provide very good data fits for the pH range 6-7, under conditions where there is a significant fraction (more than 60%) of native, intact protein in solution. For deamidation at pH 8.0, the model provides a good description of the data for the early stages of the degradation process but overestimates the fractions of monoX and monoY in late stages of the reaction. After 1 month of storage at 37 °C and pH 8.0, the fraction of native protein was around 20%, so a non-negligible fraction of doubledeamidated species was expected to be present in solution at that time. Unfortunately, the CEX method used to monitor the population of monoX and monoY species (for details see ref 3) did not allow a good separation of the double-deamidated species (doubleX, doubleY, and doubleMix); therefore, an accurate determination of the rates describing the formation of those species was not feasible. The overestimation of monoX and monoY species at late time points is a consequence that the depletion of those species by subsequent deamidation was not taken into account. The kinetic modeling performed in this case was able to predict the conversion of molecules containing IsoAsp to Asp in the hot spot. This event was confirmed by additional experiments in which Fab fragments containing either IsoAsp or Asp in the hot spot were stored under accelerated conditions: the Fab fragments with Analytical Chemistry, Vol. 82, No. 8, April 15, 2010

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IsoAsp changed in time to Asp in the hot spot, but the reverse reaction (Fab with Asp converting into IsoAsp) was not observed.3 These results suggest that, although the formation of IsoAspcontaining Fab is favored kinetically by deamidation of the hot spot, the Asp-containing Fab is more stable. In agreement with this finding, differential scanning calorimetry (DSC) results on Fab fragments present an apparent melting temperature for the Asp-containing Fab about 10 °C higher than for the IsoAsp-containing Fab.3 The same trend of Asp-containing molecules being thermodynamically favored and IsoAsp-containing molecules being kinetically favored was observed for other molecules as well,35 and it may simply reflect the disfavor of protein backbone for nonpeptidic bonds. CONCLUSIONS The kinetic modeling used to analyze the formation of deamidation products helped to predict changes occurring in the protein structure that were subsequently confirmed by additional experi(35) Capasso, S.; Di Cerbo, P. J. Pept. Res. 2000, 56, 382–387.

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ments. The examples presented in this article demonstrate that kinetic modeling can be a powerful tool for analyzing the kinetics of degradation of monoclonal antibodies. There is a large variety of kinetic schemes that can be developed to describe the degradation pathways of these complex molecules, and the purpose of this article was to illustrate the benefits of this approach through presentation of relevant examples. ACKNOWLEDGMENT We thank Dr. Henryk Mach and Dr. Yang Wang for stimulating discussions and Mrs. Lisa McCormick for editing suggestions. 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 December 3, 2009. Accepted March 4, 2010. AC902752E

Kinetics of Chemical Degradation in Monoclonal Antibodies

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