Characterization of the Acidic Species of a Monoclonal Antibody Using

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Characterization of the acidic species of a monoclonal antibody using weak cation exchange chromatography and LC-MS Gomathinayagam Ponniah, Adriana Kita, Christine Nowak, Alyssa Neill, Yekaterina Kori, Saravanamoorthy Rajendran, and Hongcheng Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02385 • Publication Date (Web): 29 Jul 2015 Downloaded from http://pubs.acs.org on August 11, 2015

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Analytical Chemistry

Characterization of the acidic species of a monoclonal antibody using weak cation exchange chromatography and LC-MS

Gomathinayagam Ponniah1, Adriana Kita1, Christine Nowak1, Alyssa Neill1, Yekaterina Kori1, Saravanamoorthy Rajendran2 and Hongcheng Liu1*

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Product Characterization and 2 Biochemical Process Developments, Alexion Pharmaceuticals Inc

352 Knotter Drive, Cheshire, CT06410

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Corresponding author

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Email: [email protected]

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Phone: 203-271-8354

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Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies have been challenging to fully characterize despite the fact that several posttranslational modifications have already been identified. The acidic species of a recombinant monoclonal antibody were collected using WCX-10 chromatography and characterized by LC-MS at multiple levels. In this study, methionine oxidation and asparagine deamidation are the only two modifications identified in the acidic species. Incubation of the collected main chromatographic peak with hydrogen peroxide generated acidic species, which confirmed that acidic species were enriched in oxidized antibody. Differences observed between the original acidic species and the oxidization-induced acidic species indicate that different mechanisms are involved in the formation of acidic species. Additionally, acidic species were generated by thermal stress of the collected main peak from the original sample. Thermal stress of the collected main peak in pH 9 buffer or ammonium bicarbonate generated chromatograms that are highly similar to those from the analysis of the original molecule. LC-MS analysis identified oxidation of the same methionine residue and deamidation of the same asparagine in the corresponding acidic fractions generated by thermal stress, however, relatively lower levels of methionine oxidation and higher levels of asparagine deamdiation were observed. The results support the use of stressed conditions to generate low abundance species for detailed characterization of recombinant monoclonal antibody charge variants, but with caution.


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Charge variation is one of the major quality attributes of recombinant monoclonal antibodies (mAbs) that has been closely monitored and tightly controlled during development. Commonly used methods to analyze charge variants include cation exchange chromatography, anion exchange chromatography, isoelectric focusing electrophoresis and capillary isoelectric focusing electrophoresis. Charge variants are classified as acidic or basic relative to the major species. Early-eluting species from cation exchange chromatography or late-eluting species from anion exchange chromatography are generally referred to as acidic species since these species, presumably, have a lower isoelectric point (pI) than the main species. On the other hand, late-eluting species from cation exchange chromatography and early-eluting species from anion exchange chromatography are referred to as basic species, as these species are presumed to have a relatively higher pI than the main species. For charge variants separated by isoelectric focusing based techniques, species with a lower pI are referred to as acidic species while those with higher pI are referred to as basic species. It is worthwhile to mention that charge variants with the same pI by isoelectric focusing techniques may have different elution times by chromatographic techniques1,2, indicating that the overall charge is not the only factor that affects chromatographic behaviors. Various modifications have been identified as the cause of the formation of either acidic or basic species. So far, the major modifications identified in acidic species include the presence of sialic acid3-7, deamidation of asparagine (Asn) residues1,2,4,5,8-11 , and glycation 5,12. The major modifications identified in basic species include the presence of partial leader sequence 11,13, uncyclized N-terminal glutamine (Gln)4,14-16, C-terminal amidation 11,17,18, succinimide formed from the isomerization of aspartate (Asp) residues 8,19-21 and the presence of C-terminal lysine (Lys)1,3,4,8,11,22. Interestingly, there are modifications that do not alter charge directly, yet could have a dramatic impact on chromatographic behaviors. For example, antibody variants without oligosaccharides or with smaller oligosaccharides23



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and antibody variants with oxidized methionine residues in the Fc region 24 are eluted off cation exchange columns as basic species. Oxidation is one of the modifications that have been thoroughly studied24-30. Two methionine (Met) residues in the Fc region are highly susceptible to oxidation. Oxidation of these residues has been reported to cause structural changes 25,28, decreased stability 28, decreased binding affinities to FcRn29,30, decreased binding to protein A 26,30and protein G26, and a shorter half-life31. As previously mentioned , a recombinant monoclonal antibody with oxidized Met residues in the Fc region appeared as basic species based on elution time when analyzed by a weak cation exchange column 24. The acidic species of a recombinant monoclonal IgG1 antibody were characterized in the current study. Fractions collected from a weak cation exchange (WCX)column were analyzed by LC-MS. Met oxidation, Asn deamidation and fragmentation are the major modifications identified in the acidic fractions. Oxidation of the same Met residue by hydrogen peroxide also generated acidic species, which supported the observation from the analysis of the original acidic species. In addition, it was found that incubation of the main peak fraction with higher pH buffers can generate acidic species profiles that are very similar to those observed from the analysis of the original mAb. The similarities and differences between the modifications identified in the acidic species generated under different conditions are further discussed.

Materials and methods Materials The recombinant monoclonal IgG1 antibody was expressed in a Chinese hamster ovary (CHO) cell line and purified at Alexion (Cheshire, CT). Acetonitrile, ammonium bicarbonate, formic acid,


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hydrogen peroxide (30%), iodoacetic acid, and trifluoroacetic acid (TFA) were purchased from Sigma (St,Louis, MO). IdeS enzyme, FabRICATOR, was purchased from Genovis (Cambridge, MA). Trypsin was purchased from Worthington (Lakewood, NJ).

Weak cation exchange chromatography A Waters Alliance HPLC and a weak cation exchange (WCX) column (ProPac WCX-10, 4.6 x 250 mm, Thermo Scientific, Sunnyvale, CA) were used for separation of antibody charge variants. Mobile phase A contained 10 mM sodium phosphate, pH 6.5. Mobile phase B contained 10 mM sodium phosphate and 500 mM sodium chloride, pH 6.5. Samples were injected at 85% mobile phase A and 15% mobile phase B. After 5 minutes, the percentage of mobile phase B was increased to 25% over 20 minutes. The column was washed using 100% mobile phase B and then equilibrated using 15% mobile phase B. The proteins eluting off the column were monitored using UV214nm and UV280nm. The flowrate was set at 1 mL/min and the column temperature was set at ambient throughout the runs. Fraction collection was guided by UV absorbance. Amicon Ultra-4 centrifuge devices (Millipore) with a molecular weight cut-off of 30 kDa were used to concentrate the collected fractions. Protein concentrations were determined using UV absorbance at 280 nm and the theoretical extinction coefficient calculated based on the known amino acid sequence.

Incubation with hydrogen peroxide The collected main peak fraction was diluted to 1 mg/mL using 10 mM sodium phosphate, pH 6.5 and incubated with 0.2%, 0.5% or 1% hydrogen peroxide at room temperature for 20 minutes. The resulting oxidized samples were buffer exchanged into 10 mM sodium phosphate using Zeba columns (ThermoScientific, Rockford, IL) and then analyzed by WCX-10 chromatography following the same procedure as previously described. Fractions corresponding to each of the separated peaks from the 5 ACS Paragon Plus Environment


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sample incubated with 0.5% hydrogen peroxide were collected and concentrated using the procedure described in the previous section. The concentrated fractions were analyzed by LC-MS after FabRICATOR digestion, which cleaves a single site in the lower hinge region of the antibody to generate F(ab’)2 and Fc fragments. In addition, the sample incubated with 1% hydrogen peroxide after buffer exchange was used for tryptic peptide mapping.

Thermal stress of the purified main peak fraction The collected main peak fraction was incubated in a buffer containing 100 mM sodium acetate, 100 mM HEPES and 100 mM Tris at pH 5, 6, 7, 8 and 9 at a final concentration of 1 mg/mL. In addition, the collected main peak fraction was also incubated in 100 mM ammonium bicarbonate. After incubation at 37 °C for 18 hours, these samples were buffer exchanged to 10 mM sodium phosphate, pH 6.5 using Zeba columns. The samples were then analyzed by WCX-10 chromatography following the same procedure as previously described. Fractions were collected from the sample stressed in ammonium bicarbonate for LC-MS analysis.

FabRICATOR digestion Fractions were diluted to 0.5 mg/mL using 10 mM sodium phosphate, pH 6.5 in a total volume of 50 µL. Each vial of FabRICATOR (5000 units) was reconstituted using 200 µL Milli-Q water. To each diluted sample, 1 µL of the reconstituted FabRICATOR was added. After digestion at room temperature for 20 minutes, the samples were analyzed by LC-MS for molecular weight determination.

LC-MS analysis of molecular weights of intact antibody and its subunits A Maxis 4 G mass spectrometer (Bruker, Billerica, MA ) and a Mass Prep MicroDesalting column (Waters, Milford, MA) were used to measure the molecular weights of the antibody and its subunits. 6 ACS Paragon Plus Environment

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Approximately 10 µg of each sample was injected into the column at 95% mobile phase A (0.1% TFA, 5% acetonitrile in water) and 5% mobile phase B (0.1% TFA in acetonitrile). After 2 minutes, the percentage of mobile phase B was increased to 90 and remained at 90% for 1.5 minutes. The column was thoroughly washed and then equilibrated with 5% mobile phase B before the next injection. Throughout the analysis, the flow-rate was set at 500 µL/min and the column temperature was set at 60 °C. The mass spectrometer was run in positive mode with the following settings: a scan range of m/z 900-5500, gas temperature of 220°C, drying gas of 10 L/min, nebulizer at 2 psig and voltage at 120eV.

LC-MS analysis of peptides Various fractions were denatured and reduced using 6 M guanidine hydrochloride in 20 mM Tris, pH 7.8 and 10 mM dithiothreitol (DTT) at 37 °C for 30 minutes. The samples were alkylated using 25 mM iodoacetic acid at 37 °C for 30 minutes. The sample pH was adjusted to approximately around 7.8 using sodium hydroxide after the addition of iodoacetic acid. The samples were then buffer exchanged into 20 mM Tris, pH 7.8 using Zeba columns (ThermoScientific, Rockford, IL). Each sample was digested using trypsin at a final trypsin to antibody ratio of 1:10 (w:w) at 37 °C for 4 hours. Tryptic peptides were analyzed using a Maxis 4 G mass spectrometer (Bruker, Billerica, MA) and an ultra-performance liquid chromatography (UPLC) system (Waters) with a Proto 200 C18 column (1.0 x 250 mm; Particle size, 10 µm; Pore size, 200A; Higgins Analytical. Inc). The samples were loaded at 98% mobile phase A (0.1% TFA in water) and 2% mobile phase B (0.1% TFA in acetonitrile). After 2 minutes, mobile phase B was increased to 35% within 118 minutes and then to 60% within 15 minutes. The column was then washed and equilibrated. The column was heated at 60 °C and the flow-rate was set at 50 µL/min. The mass spectrometer was tuned and calibrated following the manufacturer’s procedure and run at the positive scan mode with m/z in the range of 150-3000. Capillary voltage was set at 4500 V. Dry gas was set at 10 L/min. Dry temperature was set at 220 °C and the nebulizer was set at 2.0 bar. 7 ACS Paragon Plus Environment


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Results and discussion Charge variation of recombinant monoclonal antibodies is common due to various posttranslational modifications and is reflected as multiple peaks or bands detected by charged based techniques. In order to characterize those variants, fraction collection is usually a necessary step to enrich potential modifications. However, collecting a sufficient amount of low abundance species for extensive characterization is challenging. As an alternative, low abundance charge variants are often generated by stress conditions. Some stress conditions are highly specific for a particular mechanism, e.g., incubation with hydrogen peroxide to generate oxidized species. There are also conditions that are not specific and can cause degradation by multiple mechanisms, e.g., incubation of mAb at elevated temperature. It has always been a question as to whether or not the species generated by various stressed conditions are representative of the species present in the original samples. This study presents data to demonstrate the similarities and differences between acidic species generated from different conditions.

Study outlines Three major experiments were carried out in the study. In the first experiment, charge variants of a recombinant monoclonal antibody were separated by a WCX-10 column into three fractions including acidic fraction 1, acidic fraction 2 and the main peak. The fractions were characterized by LCMS for intact and subunit molecular weights and peptide mapping. Oxidation was identified as one of the major modifications in the acidic fractions. In the second experiment, the main peak fraction was oxidized using hydrogen peroxide. The oxidized samples were then analyzed by WCX-10 to determine the role of oxidation in the generation of acidic species. In the third experiment, the main peak was thermally stressed in a single buffer at various pH values or in ammonium bicarbonate. The newly generated acidic species were collected and characterized again. 8 ACS Paragon Plus Environment

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Experiment 1-Characterization of acidic species originally associated with the mAb Fraction collection

The mAb was first analyzed using a WCX-10 column. As shown in Figure 1 (Starting material), the main peak was observed at the retention time of 20 minutes. Acidic species eluting earlier than the main peak contained a region of unresolved peaks. There was also a small basic peak eluting after the main peak, however, characterization of the basic species was not the focus of the current study. The acidic species were divided into two regions. Species eluting before the small peak around 16.5 minutes are defined as acidic fraction 1. Species eluting after the small peak but before the main peak are defined as acidic fraction 2. Fractions corresponding to acidic fraction 1, acidic fraction 2 and the main peak were collected and re-analyzed by WCX-10 to confirm their purities. Minimal overlap occurred between the fractions (Figure 1). Acidic fractions 1, acidic fraction 2 and the main peak fraction were then used for LC-MS characterization. LC-MS analysis of intact molecular weights

The collected fractions were first analyzed for intact molecular weight by LC-MS to detect posttranslational modifications that are present at significant levels with substantial molecular weight differences compared to the theoretical molecular weight calculated from the known amino acids. The deconvoluted mass spectra are shown in Supporting Information Figure 1. The molecular weight of the predominant species in the main peak fraction is 147059 Da, which is in agreement with the theoretical molecular weight (147055 Da) of the mAb with G0F on both of the heavy chains. The two additional peaks correspond to the mAb with other glycoforms. The peak with the molecular weight of 147219 Da corresponds to the mAb with G0F on one heavy chain and G1F on the other heavy chain. The peak with the molecular weight of 147382 Da corresponds to the mAb with either G1F on both heavy chains or 9 ACS Paragon Plus Environment


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G0F on one heavy chain and G2F on the other heavy chain. The molecular weight of the major peak in acidic fraction 2 is 147072 Da, which is approximately 17 Da higher than the theoretical molecular weight. This 17 Da molecular weight increase indicates potential modifications such as oxidation. In acidic fraction 1, there are two major peaks with molecular weights of 147065 Da and 147075 Da, indicating potential modifications. Peaks corresponding to the mAb with G1F and G2F glycoforms on either one or both heavy chains were also observed in acidic fraction 1 and acidic fraction 2. In addition to the main peak molecular weight difference, low molecular weight fragments were also observed mainly in acidic fraction 1 and acidic fraction 2. The observation of the m/z peak series in the lower m/z range (inserts of Supporting Information Figure S1) indicates the presence of fragments. The deconvoluted mass spectra of the fragments from acidic fraction 1 are shown in Supporting Information Figure S2. Similar fragments were observed in acidic fraction 2. The peak with the molecular weight of 23483 Da, approximately 120 Da higher than the molecular weight of the light chain, is indicative of a free light chain with one cysteine modified by a free cysteine. After reduction, a peak with the exact molecular weight of the light chain was observed and thus confirmed that the peak to be a cysteinylated free light chain. The other peak with the molecular weight of 23286 Da is also related to the light chain, but its exact identity was not identified. LC-MS analysis of subunit molecular weights

Acidic fraction 1, acidic fraction 2 and the main peak were digested using FabRICATOR to generate F(ab’)2 and Fc and then analyzed by LC-MS to further localize the observed molecular weight difference. The deconvoluted mass spectra are shown in Figure 2. The molecular weight of the peak from the main peak fraction is 96626 Da, which is in agreement with the theoretical molecular weight of 96627 Da (M). Two major peaks with molecular weights of 96626 Da and 96638 Da were observed in acidic fraction 2. The observation of the higher molecular weight species indicates the existence of 10 ACS Paragon Plus Environment

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modifications. One major peak and two additional shoulder peaks with molecular weights of 96628 Da, 96641 Da and 96655 Da were observed in acidic fraction 1. Again, the presence of those peaks with higher molecular weights indicates the presence of modifications. In addition to the main peak in each spectrum, at least one additional peak with a molecular weight that is approximately 160 Da higher than the major peaks was also observed. These additional peaks probably correspond to glycation, which results in a molecular weight increase of 162 Da. LC-MS analysis of tryptic peptides

Tryptic peptides from the three fractions were analyzed to further localize the modifications identified from intact and subunit molecular weight analysis. In addition, tryptic peptides were searched for modifications with minimal molecular weight differences such as Asn deamidation, which results in a molecular weight increase of 1 Da. Modifications without a molecular weight differences such as Asp isomerization can also be detected at the peptide level because peptides containing Asp or isoAsp typically elute at different retention times. Met oxidation and Asn deamidation are the two major modifications identified by tryptic peptide mapping in acidic fractions. The sites and percentages of each modification are summarized in Table 1. Met oxidation was the major modification identified from the analysis of tryptic peptides, which is in agreement with the previous observations from intact and subunit analyses. The site of oxidation was localized to Met102 in the tryptic peptide with the amino acid sequence of GAEMTVGSWGPGTLVTVSSASTK with a monoisotopic molecular weight of 2223.1 Da (MH+). In addition, an earlier-eluting peak with a molecular weight of 2239.1 Da was observed. The molecular weight difference between the two peaks is 16 Da, corresponding to oxidation of the Met residue. This assumption was confirmed by the MS/MS spectrum (Figure 3) from fragmentation of the doubly charged ion of 1120.0 Da of the oxidized peptide. The percentage of oxidation was calculated by 11 ACS Paragon Plus Environment


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dividing the peak area of the oxidized peptide by the sum of the oxidized and native peptides and multiplied by 100. The results demonstrated 45.1% oxidation in acidic fraction 1, 22.0% in acidic fraction 2 and about 5.6 % in the main peak fraction. The level of oxidation of Met252 was also slightly higher in acidic fraction 1 (Table 1). Oxidation of this conserved Met residue has been welldocumented24-30. Deamidation is the second major modification identified in acidic fraction 1 and acidic fraction 2. An extracted ion chromatogram of the triply charged ion of the peptide with the amino acid sequence of QAPGQRPEWMGWINGGDGK is shown in Figure 4. In addition to the major peak around 75 minutes, two additional peaks eluting around 74.5 minutes and 77.8 minutes respectively were observed. Based on the triply charged m/z, the molecular weight of both the first peak and the third peak is 2085.0 (MH+), which is 1 Da higher than that of the major peak and also the theoretical molecular weight of 2084.0 (MH+). This molecular weight increase of 1 Da indicates deamidation of either an Asn residue or a glutamine (Gln) residue in this tryptic peptide. Deamidation was localized to the Asn52 by fragmentation of the triply charged ions (Supporting Information Figure S3). The percentage of deamidation was calculated by dividing the peak area of the deamidated peptide by the sum of the deamidated and native peptides and multiplied by 100. As shown in Table 1, compared to the main peak, slightly higher deamidation in acidic fraction 2 and much higher deamidation in acidic fraction 1 were observed. In summary, acidic fraction 1 contains a mixture of the mAb with oxidation of the Met residue on one heavy chain or on both heavy chains as well as higher levels of isoAsp and Asp from deamidation of Asn52 than the other fractions. Acidic fraction 2 also contains mAb with oxidation of the Met residue. A low level of modified free light chain was also observed in acidic fraction 1 and acidic fraction 2.


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Experiment 2-Characterization of acidic species generated from incubation with hydrogen peroxide A much higher level of Met oxidation was observed in the acidic fractions collected from the mAb. Other than this observation, the role of Met oxidation in the generation of acidic species remains unclear. For example, the difference between the oxidized mAb in acidic fraction 1 and the oxidized mAb in acidic fraction 2 still needs to be investigated. One possibility is that the acidic fraction 1 contains mAb with Met oxidation on both heavy chains and the acidic fraction 2 contains mAb with Met oxidation on one of the two heavy chains. Unfortunately, the evidence from analysis of the intact and subunit molecular weight is not strong enough to support the above-mentioned conclusion. Therefore, an experiment was performed by a brief incubation of the main peak with hydrogen peroxide. The short incubation time was chosen to minimize modifications other than oxidation. WCX-10 chromatograms from analysis of the main peak and the main peak incubated with various concentrations of hydrogen peroxide are shown in Figure 4. Two additional peaks eluting earlier than the original main peak were observed when the main peak was incubated with hydrogen peroxide, indicating that oxidation of the mAb can indeed cause an acidic shift on the WCX-10 column. Fractions were collected from the sample incubated with 0.5% hydrogen peroxide and analyzed by LC-MS after FabRICATOR digestion. As shown in Supporting Information Figure S4, the molecular weight of peak 1 and 2 are approximately 32 Da and 16 Da higher than the theoretical molecular weight. The molecular weight of peak 3 is in agreement with the theoretical molecular weight. Therefore, it was concluded that peak 1 contains F(ab’)2 with two oxidized Met, peak 2 contains F(ab’)2 with one oxidized Met, and peak 3 contains the non-oxidized F(ab’)2. LC-MS analysis of tryptic peptide from the oxidized material using 0.5% hydrogen peroxide demonstrated that the same Met102 was oxidized (data not shown). Interestingly, although oxidation can generate acidic species, it can only generate acidic species that eluted in the acidic fraction 2 region. It is therefore hypothesized that other additional 13 ACS Paragon Plus Environment


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modifications such as deamidation or conformational changes may have shifted the oxidized mAb to the acidic fraction 1 region. The other modifications appear to be somehow accelerated by Met oxidation.

Experiment 3-Characterization of the sample after thermal stress So far, it was demonstrated that mAb with two oxidized Met eluted earlier than mAb with one oxidized Met and both of them eluted earlier than the original main peak. However, oxidation alone is not sufficient to shift the mAb to the acidic fraction 1 region. It was proposed that additional modifications shift the oxidized mAb to acidic fraction 1. To further explore other modifications that shift oxidized mAb to the acidic fraction 1 region, the main peak fraction was incubated in different buffers or the same buffer of different pH. The second goal of this study was to demonstrate the similarities and differences between the acidic species originally associated with the mAb and the acidic species generated from thermal stress at different pH levels. The complete set of chromatograms from the analysis of the main peak after thermal stress is shown in Supporting Information Figure S5. Acidic species in the acidic fraction 2 region were generated when the main peak was incubated with buffer of pH 5, 6 and 7. Acidic species in the acidic fraction 1 region were generated from incubation of the main peak in buffers of pH 8, pH 9 and ammonium bicarbonate. Interestingly, the chromatograms obtained from the sample incubated in the pH 9 buffer or in ammonium bicarbonate are highly similar to each other and also highly similar to the chromatogram from the analysis of the original mAb (Figure 5). These highly similar chromatograms obtained from incubation of the main peak in pH 9 buffer and in ammonium bicarbonate indicate that the process is likely driven by a basic pH. The high similarity between the chromatograms of the thermally stressed sample and the chromatogram of the original sample indicate that the observed acidic charge variants may be formed by similar mechanisms.


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To further analyze the differences and similarities, fractions corresponding to acidic fraction 1, acidic fraction 2 and the main peak were collected and analyzed by following the same procedure used previously. WCX-10 chromatograms of the collected fractions are shown in Supporting Information Figure S6. Mass spectra from analysis of the intact mAb from the collected fractions are shown in Supporting Information Figure S7. Again a molecular weight higher than the calculated molecular weight was observed in the acidic fraction 1 and acidic fraction 2. The fragments were observed in the acidic fractions as demonstrated by the peak series in the relatively low m/z range shown in the inserts of supplement figure 7. The molecular weights of the fragments are shown in Supporting Information Figure S8. In addition to the two peaks corresponding to the modified light chain, other peaks were also observed. It has been demonstrated that fragmentation was accelerated at either low or high pH32 The same major modifications of Met102 oxidation and Asn52 deamidation were observed and the data is summarized in Table 2. A significantly elevated level of deamidation was observed in the acidic fraction 1 from the thermally stressed sample. In addition, a relatively lower level of oxidation was observed in the thermally stressed acidic fraction 1.

Summary As discussed previously, Met102 oxidation generated acidic species that are mainly in the acidic fraction 2 region as demonstrated by hydrogen peroxide oxidation of the collected main peak. However, acidic fraction 1 from the original mAb sample contained 45.1% oxidized Met102, while, the corresponding fraction from the thermally stressed sample contained 22.8% oxidized Met102. Assuming Asn52 deamidation was required to shift the oxidized molecules to the acidic fraction 1 region, the total amount of 30.6% deamidation (18.7%+11.9%=30.6%, see Table 2) can explain 22.8% oxidation observed in the corresponding acidic fraction 1 region in the thermally stressed sample.



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However, the total amount of deamidation of 9.8% (5.4%+4.4%=9.8%, see Table 1) cannot account for the total of 45.1% oxidation in the acidic fraction 1 of the mAb. Even if only deamidation of Asn 52 on one heavy chain was required, 9.8% deamidation can only account for 19.6%. Therefore, it can be concluded that other modifications were also involved in shifting the oxidized mAb to the acidic fraction 1 region in the original mAb. It was also hypothesized that oxidation accelerated deamidation by causing a conformational change based on the observation that both modifications were enriched in acidic fraction 1 region, while oxidation alone can only be enriched in acidic fraction 2 region. In addition, it is worthwhile to mention that the above mentioned oxidation and deamidation cannot fully account for all the acidic species. This is especially true for acidic fraction 2 in the mAb and the corresponding fraction in the thermally stressed sample.

Conclusions Acidic species of a mAb generated by three different conditions were analyzed by WCX-10 and LC-MS. The results demonstrated similarities and differences between acidic species from different conditions. It is reasonable to speculate that acidic species generated during cell culture may be formed due to several mechanisms including enzymatic or non-enzymatic reactions. Highly similar charge profiles of this mAb can be generated by simple incubation of the main peak in buffers of slightly basic pH. Detailed characterization revealed the same types of modifications at the same locations in acidic species generated during cell culture or during incubation at slightly basic pH. However, the percentage of the identified modifications varied, implying that additional mechanisms have yet to be discovered. Overall, using information generated from stressed samples to help understand acidic species associated with the mAb is very helpful, especially if the same modifications or the same mechanisms to form the acidic species can be proved scientifically.


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Associated Content Supporting Information Figure S1: Deconvoluted mass spectra of acidic fraction 1, acidic fraction 2 and the main peak. Figure S2: Deconvoluted mass spectrum of the fragments in acidic fraction 1. Figure S3: MS/MS spectra of the tryptic peptide containing either isoD, N or D. Figure S4: Deconvoluted mass spectra of F(ab’)2 from the three peaks collected from the main peak oxidized using 0.5% hydrogen peroxide. Figure S5: WCX-10 chromatograms of the original mAb, the main peak and the main peak after incubation with buffers of various pH or in ammonium bicarbonate. Figure S6: WCX-10 chromatograms of the stressed sample and its fractions. Figure S7: Devonvoluted mass spectra of the collected fractions from the stressed sample. Figure S8: Deconvoluted mass spectrum of fragments from the thermally stressed sample.



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Reference List

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Figure legends and Table titles Figure 1. WCX-10 chromatograms of the mAb. Three fractions as marked in the top panel were collected. Chromatograms from reanalysis of the collected fractions are shown for acidic fraction 1, acidic fraction 2 and main peak fraction as labeled. Figure 2. Deconvoluted mass spectra of F(ab’)2 generated from the fractions. Figure 3. MS/MS spectrum of the oxidized peptide. The amino acid sequence and the theoretical B and Y ion molecular weights are shown on top of the figures. The observed molecular weights of Y ions up to Y17 being in agreement with the calculated molecular weights indicates oxidation is on the Nterminal six amino acids, probably the Met residue. The observation of a molecular weight increase of 16 Da for B4 and other B ions confirmed oxidation of the Met residue. Figure 4. Extracted ion chromatogram of the peptides containing either isoAsp, Asn or Asp. Mass spectra of the triply charged ion from each peak are shown as inserts. Figure 5. WCX-10 chromatograms of the main peak after incubation without hydrogen peroxide or incubated with 0.2%, 0.5% or 1% as labeled in the figures. Three peaks as labled as peak 1, peak 2 and peak 3 were collected and analyzed by LC-MS after FabRICATOR digestion (Data in Supplement Figure 4) Figure 6. WCX-10 chromatograms of the main peak after incubation in the pH 9 buffer or in ammonium bicarbonate.


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Table 1. Sites and percentage of modifications identified in the original acidic fractions Table 2. Sites and percentage of modifications identified in the acidic fractions collected from the thermally stressed main peak Footnote to Table 1. The numbers represent an average of three experiments with standard deviation in the parenthesis. Footnote to Table 2. The numbers represent an average of three experiments with standard deviation in the parenthesis.



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Figure 6



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Table 1


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Table 2



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For “TOC” only


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Characterization of the Acidic Species of a Monoclonal Antibody Using

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