Protein Covalent Dimer Formation Induced by Reversed-Phase HPLC Conditions

RESEARCH ARTICLE Protein Covalent Dimer Formation Induced by Reversed-Phase HPLC Conditions XIAN HUANG, JAMES BARNARD, THOMAS M. SPITZNAGEL, RAJESH KRISHNAMURTHY Human Genome Sciences, A GlaxoSmithKline Company, Rockville, Maryland Received 9 September 2012; revised 15 November 2012; accepted 5 December 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23431 ABSTRACT: Reversed-phase high-performance liquid chromatography (RP-HPLC), which is routinely used to detect and quantitate levels of protein oxidation, was used to analyze a free cysteine-containing protein. However, the RP-HPLC method appeared to induce dimerization of the oxidized protein. The purpose of this study was to understand the role of RP-HPLC conditions in inducing protein dimerization. Samples were also analyzed by orthogonal sizebased analytical methods such as size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These methods indicated the presence of dimer and confirmed that the acidic solvent conditions induced the dimer formation of the oxidized protein. Furthermore, the dimerization was observed only when the protein was mildly oxidized and not when the protein was severely oxidized or in its native form. The sulfenic acid form of cysteine is a likely precursor to the disulfide formation. The amount of dimers increased with increasing concentration of trifluoroacetic acid (TFA) or formic acid is in the range of 0%–0.3%. The effect of the organic solvent was less than the effect of TFA/formic acid on dimer formation. Given that RP-HPLC is typically run with low-pH mobile phase containing an ion-pairing acid for improved resolution, its potential for inducing artifacts needs to be taken into consideration during method development. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: HPLC; reversed-phase; protein aggregation (dimerization); oxidation; liquid chromatography; analytical biochemistry; cysteine oxidation; cysteine sulfenic acid

INTRODUCTION In the development of well-characterized protein or polypeptide therapeutics, reversed-phase highperformance liquid chromatography (RP-HPLC) serves as an important analytical tool for formulation studies and stability indication.1,2 For example, RP-HPLC has been routinely used to detect and quantitate the amount of oxidized protein at release and upon storage.3,4 RP-HPLC methods typically use silica-based alkyl stationary phases and organoaqueous mobile phases containing trifluoroacetic acid Correspondence to: Rajesh Krishnamurthy (Telephone: +301-990-4808; Fax: +301-990-4801; E-mail: rkrishnamurthy@ zyngenia.com) Xian Huang’s present address is CMC Biologics, Inc., Bothell, Washington 98021. James Barnard’s present address is Department of Pharmaceutical Sciences, University of Colorado, Aurora, Colorado 80045. Rajesh Krishnamurthy’s present address is Zyngenia, Inc., Gaithersburg, Maryland 20878. Journal of Pharmaceutical Sciences © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association

(TFA) for gradient elution. To demonstrate or verify the capabilities of a RP-HPLC method, proteins are often oxidized using an oxidant such as hydrogen peroxide, t-butylperoxide, or t-butyl hydroperoxide prior to the sample injection.5–9 Analysis of these samples provides an indication of the number of oxidized forms, the ability of the method to resolve these species, rate of oxidation, and extent of oxidation. The site of oxidation is usually identified by analyzing fractions collected from the chromatographic profile followed by enzymatic digestion and peptide mapping.10,11 Methionine residues are often the most susceptible to oxidation.5,8,12–20 The methionine-oxidized protein is separated from its native form presenting a peak that elutes before the main peak in a typical RP-HPLC chromatogram because of the reduction of protein-surface hydrophobicity.21 On the contrary, if a protein contains a free (unpaired) cysteine (not cystine), the protein oxidation should involve cysteine oxidation because a cysteine thiol is more susceptible to oxidation than the methionine residue.16,22–29 JOURNAL OF PHARMACEUTICAL SCIENCES

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The recombinant protein we utilized in this work was albinterferon "-2b, which contained a free cysteine (Cys34) and 11 methionines.30–32 In our typical RP-HPLC chromatogram of oxidized protein, we observed not only the minor front peak representing the methionine-oxidized form, but also a small back peak, which was identified as a covalent dimer form. Surprisingly, the dimerization was not found in the oxidized sample prior to RP-HPLC, but induced by the RP-HPLC method. The purpose of this study is to confirm and understand the role of RP-HPLC conditions that induced dimerization as well as the possible mechanism of dimer formation.

MATERIALS AND METHODS Materials The recombinant protein utilized in our work was albinterferon "-2b, which is formed by genetically fusing human serum albumin to interferon "-2b. The fusion protein, which has a longer half-life compared with interferon "-2b, has a molecular weight of 88kDa with a Cys34, 19 disulfide bonds, 11 methionines and was produced in Human Genome Sciences, Inc. (Rockville, Maryland), at 5.5 mg/mL in formulation buffer (pH 7.2). The 70% (w/w) t-butyl peroxide and 30% (w/w) H2 O2 were from Sigma (St. Louis, Missouri), trifluoroacetic acid (TFA) was from Fisher (Pittsburgh, Pennsylvania), and formic acid was from Fluka (Gillingham, Kent, UK). Other reagents were from J.T. Baker (Phillipsburg, NJ). Dialysis Two types of dialysis devices from Pierce (Rockford, Illinois) were used for the buffer/solvent exchanges: R dialysis cassettes, 3500 molecu(1) Slide-A-Lyzer lar weight cut-off (MWCO) for 3–12mL sample volR MINI dialysis units, 3500 ume and (2) Slide-A-Lyzer MWCO for sample volume 10–100 :L. Oxidation Albinterferon "-2b was oxidized by the following two methods in parallel: (1) A certain amount of protein (e.g., 5 mL of 5.5 mg/mL protein in formulation buffer at pH 7.2) was treated with t-butyl peroxide at an initial concentration of 0.03% (w/v) at 5◦ C for 24 h and (2) 5 mL of the same protein solution was treated with hydrogen peroxide at an initial concentration of 0.003% (w/v) at 5◦ C for 1h. The choice of oxidants and concentration were based on conditions used in forced oxidation studies with other proteins. Each sample was R dialysis cassette then dialyzed with a Slide-A-Lyzer (3500 MWCO) against two 500 mL formulation buffer exchanges to replace the oxidizing buffer and to stop the forced oxidation. Oxidized samples were stored in 500:L aliquots at −80◦ C until use. The samples preJOURNAL OF PHARMACEUTICAL SCIENCES

pared by the second (hydrogen peroxide) method were used only for the oxidation pathway study. Incubation in Acidic Solvents To simulate the RP-HPLC mobile phase conditions and to investigate their effect on albinterferon "-2b dimerization, solvents containing 0, 20, 40, or 60% (v/v) acetonitrile (ACN) in water and 0.05, 0.1, 0.15, or 0.3% (v/v) TFA or formic acid were prepared. Each oxidized or untreated (control) sample (100:L) was dialyzed against the different solvents for 45 minutes typically at room temperature (about 25◦ C). Then, each sample (in an incubating solvent) was dialyzed against (back to) formulation buffer prior to size-exclusion high-performance liquid chromatography (SE-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. For each incubated sample, the RP-HPLC was also performed to detect the dimer content. Size-Exclusion High-Performance Liquid Chromatography SE-HPLC was performed on Waters 2690 Alliance HPLC system (with Waters 2996 photodiode array (PDA) detector) using a Tosohaas G3000SWXL column (7.8 × 300 mm; Tosoh, Grove City, Ohio) at room temperature. Isocratic elution of the buffer containing 104 mM sodium phosphate and 100mM sodium sulfate (pH 6.7) was carried out at a flow rate of 1.0 mL/min for 20 min. Detection was performed at UV 280 nm. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Novex 4%–12% bis–tris precast polyacrylamide gels and relevant reagent kits from Invitrogen (Carlsbad, California) were used to perform SDS-PAGE. Unless otherwise noted, the Novex precast gel electrophoresis guide was used to design the sample preparation and SDS-PAGE experiments. Both reduced and nonreduced protein samples were prepared using the NuPAGE reagent kit. Dithiothreitol in the reagent kit was used for the disulfide reduction. Reduced samples were prepared using a 25:20:5 ratio of the NuPAGE lithium dodecyl sulfide sample buffer–test sample–NuPAGE 10× reducing agent. Nonreduced samples were prepared using a 25:20:5 ratio of NuPAGE lithium dodecyl sulfide sample buffer–test sample–water for injection. Test sample concentration was adjusted to allow for a 4 :g load at a 25 :L load volume. Both nonreduced and reduced samples were heated at 95◦ C for 2min. SDS-PAGE was performed at a constant voltage of 200V for 35min. The resulting gels were stained for 1h with Invitrogen Simply Blue, followed by 2h destaining in deionized water. Densitometry was performed using a DOI 10.1002/jps

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Figure 1. Chromatograms of albinterferon "-2b before and after the forced oxidation. (a) Direct SE-HPLC did not detect changes in dimer amount for the oxidized sample; (b) RP-HPLC resulted in an increased back peak, which was subsequently identified as dimer by fraction collection followed by LC–ESI/MS and SE-HPLC (data not shown).

Personal Densitometer SI with ImageQuant software (GE Healthcare, Waukesha, Wisconsin). Reversed-Phase High-Performance Liquid Chromatography Samples were analyzed on Waters 2690 Alliance HPLC system (with Waters 2996 PDA detector) using a Phenomenex Jupiter C4 column (4.6 × 250 mm; ˚ Torrance, California). Mobile phase used 5 :m, 300A; for gradient elution: A = 0.1% (v/v) TFA in water and B = 0.1% (v/v) TFA in ACN. The elution program comprised a flow rate of 0.5 mL/min with linear gradient from 42% B to 58% B over a period of 32 min, a flow rate of 1.0 mL/min at 80% B for 6min, and a flow rate of 0.5 mL/min at 42% B for 16min. The column temperature was controlled at 50◦ C. Detection was performed at UV 215 nm.

RESULTS AND DISCUSSION Dimer Formation Induced by RP-HPLC In forced oxidation studies with 0.03% (w/v) t-butyl peroxide at 5◦ C for 24 h, the RP-HPLC method was able to separate the methionine-oxidized form from its native protein, presenting a minor peak before the main peak in the elution chromatogram. Meanwhile, a minor back peak was also observed as shown in Figure 1b. The back peak was identified as a dimer form by RP-HPLC–mass spectrometry (MS) as DOI 10.1002/jps

well as SE-HPLC when a fraction collected from the RP-HPLC back peak was analyzed. This was surprising, considering that the initial starting material did not contain any dimers. It appeared to suggest that oxidizing albinterferon "-2b was inducing dimerization. However, this possibility was eliminated when samples before and after oxidation were analyzed by SE-HPLC (a conventional and orthogonal technique to RP-HPLC for detecting dimers and aggregates). The SE-HPLC chromatograms (Fig. 1a) of both the oxidized and the intact samples were similar and did not indicate the presence of dimers. The SE-HPLC method is capable of detecting dimers, as demonstrated in Figure 4. These studies implied that albinterferon "-2b dimers detected by RP-HPLC did not exist in the original oxidized sample but were induced by the RP-HPLC conditions. All of the observations are summarized in Figure 2. For the oxidized protein, dimer form induced by the RP-HPLC with 0.1% TFA was detected by subsequent (1) online electrospray ionization mass spectrometry (ESI–MS), (2) SE-HPLC, and (3) reduced/ nonreduced SDS-PAGE. Taking all of these observations (Fig. 3) into consideration, we concluded that the dimeric form did not form immediately upon oxidation, the RP-HPLC conditions were causing dimerization of the oxidized protein, and that the RP-HPLC method did not “indiscriminately” induce an artifact; the dimer form JOURNAL OF PHARMACEUTICAL SCIENCES

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Figure 2. Confirmation of the RP-HPLC-induced dimer of the oxidant-stressed protein.

appeared only when the protein sample was pretreated with an oxidizing agent. Dimerization Induced by Simulated RP-HPLC Solvent Conditions To determine whether the dimer was formed because of the mobile phase, the oxidant-treated protein was incubated in water or water–ACN solvents containing TFA or formic acid followed by analysis with SEHPLC and SDS-PAGE. It should be noted that the oxidized protein was only incubated in the acidic solvent without going through the RP-HPLC column and system. Thus, this solvent incubation approach

was performed to simulate HPLC mobile phase conditions. Since both the intact and oxidized protein samples contained buffer with high salt concentration, the acidic solvent incubation started practically with buffer/solvent exchange, that is, the sample in its buffer was dialyzed against the large quantity of acidic solvent. The dialysis (incubation) was performed at room temperature for 45 min for each sample. We had observed earlier that the solvent temperature in the range of room temperature to 50◦ C was not a sensitive factor to the dimer formation and dimer amount. Thus to simplify the experiments,

Figure 3. Scheme of investigations and observations concerning the dimer formation under acidic solvent conditions. JOURNAL OF PHARMACEUTICAL SCIENCES

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Figure 4. Effect of TFA concentration on the dimer formation in aqueous solutions as measured by SE-HPLC (a and b) and reconfirmed by RP-HPLC with 0.1% TFA in mobile phase (c). Samples: the t-butyl peroxide-treated protein and intact protein (control) in formulation buffer.

dialysis and incubation were performed at room temperature. The investigations and observations concerning the dimer formation under acidic solvent conditions are schematically summarized in Figure 3. As indicated in Figure 3, the control and oxidized proteins were dialyzed against different levels of TFA or formic acid in different mixtures of ACN and water prior to the dimer measurement. The results corresponding to these samples are shown in Figure 4 through Figure 7. Figure 4 shows the effect of TFA concentration on the dimer formation in aqueous solutions as measured by SE-HPLC and reconfirmed by RP-HPLC with 0.1% TFA in mobile phase. As measured by SEHPLC following the acidic solvent incubation with 0%–0.3% TFA, there was no measurable dimerization from the nonoxidized protein, whereas the incubation led to significant dimerization of the t-butyl-

peroxide-treated protein. Further, the induced dimer amount increased with increasing TFA concentration. RP-HPLC analysis of these samples also yielded similar results, that is, there was little evidence of dimerization with the unoxidized samples despite exposure to TFA. The oxidized sample exhibited dimerization that increased with increasing TFA concentration. Figure 5 depicts the relationship between induced dimer amount against TFA or formic acid concentration in aqueous solutions as measured by SEHPLC and RP-HPLC. For the oxidized protein, the data demonstrate that the induced dimer amount increased significantly with the acid concentration from 0% to 0.15%, whereas between 0.15% and 0.30% acid, there was no significant increase in dimer. For intact or nonoxidized protein, as expected, the acids did not induce dimerization. Figure 6 shows the effect of ACN on the dimer formation in the oxidized samples as measured by

Figure 5. Effect of (a) TFA or (b) formic acid concentration on the dimer formation in aqueous solutions as measured by SE-HPLC and RP-HPLC. Samples: the t-butyl peroxide-treated protein and intact protein, both in formulation buffer. DOI 10.1002/jps

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Figure 6. Effect of (a) TFA or (b) formic acid concentration on the dimer formation in aqueous or organo-aqueous (40% ACN) solution. The dimer amount was measured by SE-HPLC from both intact and the t-butyl peroxide-treated samples.

SE-HPLC. The influence of 40% ACN on inducing dimerization can be observed by comparing the curves describing exposure to TFA/formic acid alone and those describing the effect of exposure to TFA/formic acid in 40% ACN. The data indicate that the presence of 40% ACN induced greater dimerization of albinterferon "-2b in the oxidized samples. This could be explained by the denaturing effect that organo-aqueous solutions have on proteins, that is, dimerization may be accelerated as a result of the denaturation of the protein. In addition, the identity of the ion-pairing agent did not appear to play a very significant role in minimizing dimerization because TFA and formic acid exhibited similar rise in dimer level with concentration. The results in Figures 5 and 6 also indicate that the dimerization induced by an acid was fast and can occur within a few minutes. In this acidic solvent incubation study, the duration of exposure of the protein to the acidic solvents was less than 45 min. A typical protein–solvent contacting time in which a sample passes through injection point to the post-column detection point in RP-HPLC appears sufficiently long enough to induce dimer formation. The nonreduced and reduced SDS-PAGE analyses of the protein samples incubated with TFA or formic

acid in water or 40% ACN are shown in Figure 7, where, in all cases, the top band (dimer) and bottom band (monomer) of the nonreduced samples yielded monomer after reduction. The results clearly indicated that the dimer form was disulfide mediated. It also implied that the Cys34 thiol group ( SH), the only free cysteine in the protein, was likely involved in the covalent dimer formation. Oxidation Pathway and the Intermolecular Dimerization Mechanism In this study, because the dimer is formed by disulfide bonding (Fig. 7), albinterferon "-2b oxidation likely involved the oxidation of the Cys34 thiol (Cys SH) producing cysteine sulfenic acid (Cys S OH).33–35 We believe that this intermediate upon exposure to the acidic mobile phases typically used in RP-HPLC leads to protein dimerization via disulfide bonds. Unlike small molecules with unpaired thiols that are oxidized to sulfenic acid and immediately form intermolecular disulfides,34,36 a protein containing a Cys34 thiol may have more complicated oxidation pathways that could lead to multiple products29,37 as depicted in Figure 8. For example, the protein Cys34 thiol oxidation may not immediately produce intermolecular disulfides (covalent dimers) because of

Figure 7. SDS-PAGE of nonreduced (NR) and reduced (R) samples from incubation in acidic solvents indicated that the dimer form was reducible with. Acidic incubation conditions: 0.05% and 0.30% TFA or formic acid in water or 40% ACN at room temperature. JOURNAL OF PHARMACEUTICAL SCIENCES

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Figure 8. Proposed protein Cys34 thiol oxidation pathway.

steric hindrance that limits the cross talk between an oxidized thiol and a second thiol.29 In this case, the cysteine–sulfenic acid becomes a nucleophilic intermediate that can be unusually stable in certain proteins as described by Claiborne et al.38,39 As an important model, human serum albumin, which contains a Cys34, has been intensively studied and known to form a remarkably stable intermediate containing cysteine–sulfenic acid.40,41 Specifically, the only approximately 15% of the sulfenic acid in human serum albumin was found to decay after 2h at 37◦ C under aerobic conditions.40 In general, depending on environment as well as the size and structure of a Cys34containing protein, its cysteine–sulfenic acid may provide a metastable oxidized form or otherwise give rise to more stable disulfide, sulfinic acid, or sulfonic acid forms.25,37,42,43 To further investigate the relationship between the oxidation of the Cys34-containing protein and the subsequent dimer formation induced by the RPHPLC conditions, albinterferon "-2b was exposed to 0.003% (w/v) hydrogen peroxide at 5◦ C for varying times (sampling at 1, 2, . . ., 10h) followed by RP-HPLC analysis with 0.1% TFA in mobile phase (Fig. 9). We observed that the amount of dimer varied on the basis of the duration of exposure to the oxidant. The

dimer level initially increased rapidly, remained relatively constant for a short duration, and then decreased with extended exposure to the oxidant. This behavior is usually observed when an intermediate is involved and when the concentration of the intermediate that induces dimerization increases initially and then decreases with time (Fig. 8). Observations from several publications25,29,37,39–43 appear to provide sufficient basis to attempt to explain our observations. These studies had demonstrated the possibility of dimer formation from the sulfenic acid intermediate, but not the sulfinic or cysteic acid form which cannot lead to disulfide products.40 As Claiborne et al.39 reported, mild or limited oxidation could yield a stable sulfenic acid form. We believe that the oxidation with 0.03% t-butyl peroxide at 5◦ C for 24 h was suitably mild, resulting in a relatively stable oxidized protein intermediate, the cysteine sulfenic acid form. This intermediate led to dimer formation subsequently upon contacting 0.1% TFA in RP-HPLC elution, as shown in Figures 5 and 6. Similarly, in the oxidation and dimerization relationship study (Fig. 9) with the very low concentration of hydrogen peroxide, we reason that the oxidation produced a stable sulfenic acid form initially. When the exposure to the oxidant continued,

Figure 9. Direct RP-HPLC runs of albinterferon "-2b samples treated with hydrogen peroxide at 0.03% (v/v) for different times. Mobile phase for gradient elution contained 0.1% (v/v) TFA. (a) Dimer content induced and detected by RP-HPLC of samples from oxidation pulls; (b) Sequential chromatograms corresponding to three representative data points in (a). DOI 10.1002/jps

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the sulfenic acid (Cys S OH) form converted to the irreversible sulfinic acid (Cys SO OH) or sulfonic acid (Cys SO2 OH) form.42 In the process, the concentration of the cysteine sulfenic acid intermediates decreased so that the amount of dimer formed is reduced. This model is supported by the results from RP-HPLC of the oxidation samples corresponding to different oxidation times as shown in Figure 9. Studies conducted to better understand oxidative protein folding in vivo44–47 suggest that oxidation involving peroxides kinetically favor disulfide bond formation over the oxidation of cysteine to cysteine sulfinic acid unless the peroxide amount were in excess, which is consistent with our observations. Further, these studies also indicated that the sulfenic acid form could revert to the reduced thiol state leading to the formation of disulfide bonds with other free thiols. In this study (Fig. 9), the protein Cys34 thiol was exposed to two different conditions: mild oxidation at a pH above neutral (pH 7.2) followed by exposure to acidic mobile phase in RP-HPLC at a very low pH. As already confirmed, the disulfide dimers formed only in the second step (the RP-HPLC run). This observation implies that neutral or higher pH may not induce the sulfenic acid intermediate to form disulfide products during oxidation.40 This could be because of steric hindrance that prevent two large molecules from forming a dimer via disulfide bonding, as explained by Griffiths et al.29 However, when the pH is significantly reduced and in the presence of organic solvent, potentially denaturing the protein, the steric hindrance to disulfide formation may be overcome. The denaturation of the protein at low pH was confirmed by circular dichrosim (data not shown). Denaturation at low pH has also been demonstrated for several other proteins.38–40 Meanwhile, the low pH could also cause the cysteine sulfenic acid to become unstable or react with a thiolate ion to form disulfides.43 Our observations indeed suggest that acidic or low-pH conditions (like that provided by RP-HPLC mobile phase) tend to induce disulfide-mediated dimerization when cysteine sulfenic acid is present. Support for the proposed mechanism would be strong if the three different forms of the Cys34 oxidized protein could be detected and identified by an analytical technique such as mass spectrometry. As this protein has one Cys34 and 11 methionines, its oxidation product can be highly heterogeneous, and ESI–MS did not resolve the heterogeneous oxidized monomer forms when analyzed as an intact protein. Theoretically, nonreduced peptide mapping by LC–MS may detect the Cys34–Cys34-linked peptide to confirm the covalent dimer. However, the detection of other three Cys34-containing peptides with Cys SOH, Cys SOOH, or Cys SOOOH may not be achieved. A lengthy protein digestion (e.g., 37◦ C for 18h) prior to LC–MS analysis could induce JOURNAL OF PHARMACEUTICAL SCIENCES

artifacts, making unambiguous support for the mechanism highly challenging. A model peptide (say a short peptide containing a Cysteine but no methionine) could be utilized to study the formation of the intermediate and the proposed mechanism and is a subject for future investigations. In summary, we believe that our observations possess important implications for the design of stressed oxidation studies as well as the interpretation of RP-HPLC data when dealing with proteins containing Cysteine. As our results demonstrate, there is the potential to be misled into thinking that dimers have been formed because of oxidation. This would cause the overall purity of the molecule, estimated by RP-HPLC, to be lower than the true value. It also underscores the importance of having orthogonal methods (SEC in our case) while establishing the degradation pathway of the protein. When a free cysteine is present, the conditions of forced oxidation studies (choice and level of oxidizing agent) should be carefully explored because different forms of oxidized cysteine can lead to multiple end products of the reaction. As previous publications29,36–41 point out, it is likely that these cysteine forms are relatively stable when they occur in protein compared with similar studies performed on small molecules. The ionpairing agent is typically added to improve the resolution between different variant forms. As one considers improving resolution to be an important objective in optimizing the method, increasing concentrations of the ion-pairing agent would seem to be a natural choice in achieving this objective. However, the concentration of the ion-pairing agent can influence the amount of dimerization, and hence, its level needs to be selected after due investigation.

CONCLUSIONS The dimer observed during forced oxidation studies is formed as a result of solvent conditions used for RP-HPLC and is linked by disulfide bonds. Only mildly oxidized protein leads to the dimerization, whereas both intact and overoxidized forms do not lead to the dimerization. The addition of an acid (e.g., 0.1% TFA or formic acid) and the presence of organic solvent in the mobile phase for RP-HPLC are major causes for the induced dimer of the oxidized protein. The amount of dimer induced is acid-concentration dependent. Addition of organic modifier increases the dimer content but is not the primary factor causing dimerization. For the Cysteine-containing protein samples, it is prudent to verify that the conditions used for RP-HPLC do not induce dimerization, especially when the protein is mildly oxidized during the conduct of stressed studies or upon storage. DOI 10.1002/jps

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DOI 10.1002/jps