Characterization of Drug-Product-Related Impurities and Variants of a

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Characterization of Drug Product-related Impurities and Variants of a Therapeutic Monoclonal Antibody by Higher-energy C-trap Dissociation Mass Spectrometry Deyun Wang, Colin Wynne, Flora Gu, Christopher Becker, Jia Zhao, Hans-Martin Mueller, Huijuan Li, Mohammed Shameem, and Yan-Hui Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503158g • Publication Date (Web): 16 Dec 2014 Downloaded from http://pubs.acs.org on December 20, 2014

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Analytical Chemistry

Characterization of Drug Product-related Impurities and Variants of a Therapeutic Monoclonal Antibody by Higher-energy C-trap Dissociation Mass Spectrometry Deyun Wang,++ Colin Wynne,++ Flora Gu,+ Chris Becker ,+++ Jia Zhao, + Hans-Martin Mueller, Huijuan Li,+ Mohammed Shameem,+ and Yan-Hui Liu+*

+

AUTHOR ADDRESS +

Protein Mass Spectrometry, Sterile Product and Analytical Development, Bioprocess

Development, Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA ++

Eurofins-Lancaster Laboratories Inc., 2425 New Holland Pike, Lancaster, PA 17601, USA

+++

Protein Metrics Inc., 1622 San Carlos Ave., Suite C, San Carlos, CA 94070, USA

*Corresponding author. E-mail: [email protected]

ACS Paragon Plus Environment

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KEYWORDS Middle-down LC/MS-MS, HCD, Q-Exactive Orbitrap MS, ESI, therapeutic monoclonal antibody, anti-C. diff monoclonal antibody, IgG1, drug product-related impurities and variants, post-translational modification


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Abstract

Mass spectrometry (MS) characterization of recombinant monoclonal antibody (mAb) drugs and their degraded and/or post-translationally modified counterparts, drug product-related impurities and variants, is critical for successful development of the biotherapeutics. Specifically in this study, drug product-related impurities of an anti-Clostridium difficile (C. diff) IgG1 mAb drug substance (DS) were profiled by cation-exchange liquid chromatography (CEX) followed by the CEX peaks being fraction-collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methodology is developed on a Thermo QExactive Orbitrap MS for (1) accurate mass measurements of the mAb, its CEX fractionated impurities, and their respective heavy chains and light chains; and (2) middle-down LC/MS-MS of the light chains and the heavy chains using higher-energy C-trap dissociation (HCD). The accurate mass measurements and the HCD middle-down MS/MS experiments identify that major impurities and variants of the anti-C. diff mAb are degradation species of the heavy chains at residue Asn101 as well as at the hinge region amino acids including Cys222, Lys224, His226 and Thr227, with levels ranged from 0.3% to 6.2% of the total drug substance. Additional impurities were identified as light chain C-terminal truncation at Gly93 and oxidized heavy chains at Met40, Met93 and Met430. Our impurity characterization results demonstrate that the middle-down MS method allows direct and accurate identification of drug product-related impurities of therapeutic mAbs. It is particularly useful for those low-level impurities and variants that are not suitable for further fractionation and characterization by bottom-up MS.


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Introduction Biologics such as recombinant monoclonal antibodies (mAbs) are a fast growing sector in the current pharmaceutical market.1, 2 Drug product-related impurities and variants of a therapeutic mAb, generated from product degradation, post-translational modifications (PTMs), and/or chemical modifications, are of great importance in the drug development because of the impurities’ potential immunogenicity in the clinic.3 Therefore detailed characterization of drug product-related impurities and variants are required by the Food and Drug Administration (FDA).4 Mass spectrometry (MS) has been essential in impurity characterization in pharmaceutical industry because it can provide specific information of a molecule’s primary sequence information including modifications with a wide dynamic range in detection.5 In general, MS-based structure characterization of proteins includes the “bottom-up”, and the “topdown” or the “middle-down” approaches.6, 7 In the bottom-up approach, a protein is enzymatically digested 8 and/or chemically cleaved into peptides, followed by liquid chromatography (LC)/tandem MS analysis in a data-dependent or data-independent manner, or by MALDI MS(/MS) analysis, to obtain molecular masses and sequence information of the peptides. In most cases, this approach is highly sensitive for identification of sequences of the peptides of a protein including PTMs or chemical modifications on a variety of mass spectrometry instruments.9, 10 Recent improvement in MS instrumentation, such as the Orbitrap, further facilitates the identification of these modifications, e.g. iso- aspartic acid can be identified by online LC/MS-MS without the need for Edman degradation reaction.11 High sequence coverage of the proteins including therapeutic monoclonal antibodies 12 is usually obtained by bottom-up, especially when multiple enzyme digestion is applied. However, the information of the protein as an entity could be lost by the bottom-up approach. In addition, this approach could


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be time consuming and may introduce artificial modifications during sample handling.13-15 With the advances in MS instrumentation and software developments, top-down and middle-down MS has been increasingly applied in proteomics and in recombinant protein characterizations for drug discovery and development.16 Top-down MS involves measurement of a protein’s molecular mass followed by direct gas-phase dissociation of the molecule without digestion. Analogically, the middle-down includes the MS/MS analysis of protein subunits from limited proteinase digestion, such as papain 11 and IdeS,17 or chemical cleavage. Direct fragmentation of protein molecules or their subunits are traditionally performed by collision-induced dissociation (CID),18-20 electron transfer dissociation (ETD),21 electron capture dissociation (ECD),22 and ultraviolet photodissociation (UVPD) techniques.23-27 CID has been shown to generate limited fragmentation on intact antibodies,28 the light chains and the heavy chains of mAbs 29, 30 or small proteins with similar molecular weight 31 The ECD and ETD are more efficient on protein fragmentation than CID.32-34 While ECD experiments are usually conducted on a FT-ICR MS instrument,35-38 ETD, in contrast, has been widely applied on various types of mass spectrometers for protein characterization.21, 39 Recently, ETD has also been used for top-down MS/MS of intact mAb (see Table S3),21, 40 and for the middle down analysis of antibody subunits from limited enzyme digestion 17 or DTT-reduction (see Table S3).41 Tsybin et al reported a 33% sequence coverage of a Humira IgG1 in the ETD top-down experiments of the intact mAb molecule.40 More recently, Fornelli reported the IdeS-assisted middle-down on the subunits of IgG1 antibodies by ETD. The sequence coverage achieved is about 30%~50% for the light chains, Fd and part of Fc subunits with a single LC/MS/MS run, and was improved to 60%~70% after combining and averaging the data from 4 to 10 LC runs.17 In comparison with CID and ETD, UVPD is recently shown to be more efficient in the top-down MS/MS analysis of


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proteins or complexes up to 30 kDa.23, 24, 27 However, no UVPD for mAb top/middle-down analysis is reported up to the time this manuscript is submitted. For structural elucidation, higher-energy C-trap dissociation (HCD) was developed to perform in an octopole collision cell of an Orbitrap mass spectrometer for gas phase dissociation of peptides and proteins.42 It has been shown to be more efficient for protein dissociation than CID.43 Reports have been published on the HCD middle-down MS/MS analysis of antibody subunits from proteinase-digestion or DTT-reduction (see Table S3),43 and top-down MS/MS on intact proteins with molecular masses less than 47 kDa.44, 45 In this study, we present a HCD middle-down MS/MS method for the characterization of lowlevel drug product-related impurities and variants presented in a stability sample of a recombinant anti-Clostridium difficile (C. diff) mAb drug substance. This immunoglobulin G1 (IgG1) monoclonal antibody is developed to target the C. diff Toxin B for the treatment of C. diff infection.46 Its drug product-related impurities and variants, generated from a three-month accelerated stability study of the mAb under 40 °C and 75% relative humidity condition (RH4), are profiled and fraction-collected by cation exchange chromatography (CEX). For structural characterization, the monoclonal antibody and its collected impurity fractions are subjected to LC/MS for mass measurements on a Thermo Q-Exactive Orbitrap mass spectrometer, where molecular masses and information of overall protein heterogeneity in each CEX fraction are obtained.47 Even though CEX is applied to separate the impurities, multiple impurity species are found to co-exist in several CEX fractions. Given the low level impurities and variants in the mAb drug substance (0.3%~6.2%) and the need for complete sequence characterization, especially for mAb fragments, further fractionation of those impurities and variants for bottomup MS characterization would be more material- and time-consuming. A middle-down


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LC/tandem MS method by HCD was thus applied on a Q-Exactive Orbitrap MS for structural elucidation of the low-level and site specific modification impurities of the anti- C. diff mAb. The middle-down tandem MS method proved to be capable of providing comprehensive sequence coverage map of the light and the heavy chains of anti-C. diff mAb, as well as to identify the drug product-related clipping impurities and variants. Materials and Methods The reagents and CEX fractionation of Anti-C. diff mAb can be found in the Supporting Information. LC/ESI-MS of Intact mAb Online LC separation was conducted on a Waters Acquity UPLC H-class system (Waters, Milford, MA) by using a BEH300 C4 column (1.7 µm, 1.0 × 100 mm), with mobile phase A being 0.1% formic acid (FA) in water and mobile phase B being 0.1% FA in acetonitrile (ACN). The LC flow rate was 0.08 mL/min and the column temperature was maintained at 80 ºC. The antibody was eluted using a gradient of 30.0% – 90.0% B in 4.0 – 15.0 min. MS spectra were acquired on a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) in centroid mode with a resolution of 17,500 at m/z 400. Multiple charge states of the proteins were obtained in m/z range of 600 – 4000. The MS instrument settings were as follows: ion spray voltage of 4.5 kV, in-source CID voltage of 50 V, S-lens level of 65, sheath gas flow rate of 22, auxiliary gas flow rate of 5 and capillary temperature at 275°C. The Q-Exactive Orbitrap was operated with an AGC target setting of 3e6 and a maximum IT setting of 250 ms for 10 microscans.


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LC/ESI-MS of Disulfide Bond-reduced Anti-C. diff mAb and Its Product-related Impurities Fractionated by CEX Anti-C. diff mAb stability sample and its CEX fractions were reduced by 20 mM DTT in a buffer containing 50 mM Tris (pH 8.0) and 6 M guanidine hydrochloride. The reaction was conducted at 56 º C for 20 min. LC/MS was performed on a Waters Acquity UPLC H-class system coupled on-line with a Thermo Q-Exactive Orbitrap MS. Waters BEH C4 column (2.1 × 100 mm, 1.7 µm) was applied for separation with mobile phase A as 0.05% trifluoroacetic acid (TFA) in water and mobile phase B as 0.04% TFA in ACN. The column temperature was maintained at 60 ºC. The separation gradient was 31.0% – 34.0 % B in 3.0 – 24.0 min with a flow rate of 0.2 mL/min. The wavelength of UV detector was set at 214 nm. MS spectra in centroid mode were scanned in a range of m/z 600 – 4000 with a resolution of 17,500 at m/z 400. MS instrument settings were: ion spray voltage of 4.0 kV, in-source CID voltage of 10 V, S-lens level of 55, sheath gas flow rate of 22, auxiliary gas flow rate of 5, and capillary temperature at 275°C. The Q-Exactive Orbitrap was operated with an AGC target setting of 3e6 and a maximum IT setting of 250 ms for 10 microscans. HCD Middle-down LC/MS-MS of Disulfide Bond-reduced Anti-C. diff mAb and Its Product-related Impurities Fractionated by CEX The DTT-reduced samples were separated using the HPLC conditions that was used for reduced mass analysis, followed by HCD MS/MS on the Q-Exactive Orbitrap MS. The MS conditions applied were: ion spray voltage of 4.0 kV, in-source CID voltage of 10 V, S-lens level of 55, sheath gas flow rate of 22, the auxiliary gas flow rate of 5, and capillary temperature at 275°C. The Q-Exactive Orbitrap was operated with an AGC target setting of 1e6 and a maximum IT


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setting of 200 ms for 10 microscans. The precursor ions of a protein molecule were selected for HCD MS/MS with an isolation window of m/z 5. The mass resolution was set at 140,000 (at m/z 400) for MS/MS. MS/MS was acquired in a range of m/z 250 to 4000. The normalized collision energy (NCE) for each individual charge state of a protein was optimized in a range of 18% – 30% (30.7 eV – 105.5 eV). In case of simultaneous gas-phase dissociation of multiple charge states of a protein, the optimized collision energy of each charge state was applied for the corresponding precursor ion. The AGC target was set at 1e6 with a maximum IT set of 250 ms for 10 microscans. In a pseudo MS3 experiment through in-source CID followed by HCD MS/MS, the in-source CID was increased to 45 V to fragment the heavy chains of the mAb, and a resulted fragment ion was subsequently isolated for HCD MS/MS using the same MS instrument conditions as above. MS Data Analysis The MS data from intact mass and reduced mass experiments were deconvoluted by Protein Deconvolution 1.0 (Thermo Fisher Scientific, San Jose, CA) to obtain average masses of the intact mAb and the heavy chains, as well as to obtain monoisotopic masses of the light chains and the anti-C. diff impurities with molecular masses < 25 kDa. The middle-down MS/MS raw data files of the light chains, the heavy chains and the impurities were processed by ProSight PC 3.0 (Thermo Fisher Scientific, San Jose, CA) with a mass tolerance of 10.0 ppm. The light chain data was additionally processed by Byonic (Protein Metrics, San Carlos, CA) with a mass tolerance of 10.0 ppm and 1% FDR. Furthermore, all the fragment ion assignments were manually inspected. Results and Discussions


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Mass Measurements of an RH4 Stability Sample of Anti-C. diff mAb and Its Productrelated Impurities Fractionated by CEX The anti-C. diff mAb RH4 stability sample as well as each of its CEX fractions were investigated by LC/MS for intact mass analysis and overall evaluation of molecular heterogeneity. Intact IgG1 molecule is detected in the RH4 stability sample and in its CEX Fractions 3 – 10. The individual glycoforms of the intact mAb,48 e.g. with G0, G0F, G1F and G2F on each of the heavy chains, are baseline resolved in individual charge states from 44+ to 72+ as shown in Figure 1(A). The average mass value of 148200.36 Da is detected for the G0 glycoform of the intact anti-C. diff mAb (Figure 1(B)), which agrees with its theoretical average molecular mass of 148200.94 Da with a mass measurement accuracy of 3.9 ppm. Besides the intact mAb, six additional masses are also detected in CEX Fractions 1- 5 as shown in Table S1 with molecular masses range from 11 kDa to 48 kDa for Impurities 2 to 7. In CEX Fractions 6 -10, only intact molecular species is detected. Since all of the 7 impurities are presented in either CEX Fraction 4 or Fraction 5, these two CEX fractions are further characterized in detail by reduced mass measurements and by HCD middle-down MS/MS. DTT treatment of anti-C. diff mAb and its impurities and variants reduced the inter- and intrachain disulfide bonds to generate heavy chains and light chains with reduced sizes for MS characterization. As summarized in Table S2, in addition to the expected heavy chain with a G0F N-glycosylation and C-terminal lysine truncation (mass measurement accuracy of - 4.5 ppm; data for heavy chain with a G1F N-glycosylation is not shown) and the light chain (mass measurement accuracy of 1.6 ppm), an additional twelve molecular species are detected in the DTT-reduced CEX Fraction 4 (Figure S1) and Fraction 5 (Figure S2). As shown in Figure S1, the isotopic peaks of the major charge state (10+) of the light chains as well as of the impurities


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with molecular mass less than 30 kDa are well resolved with a resolution setting of 140,000 at m/z 400, leading to monoisotopic mass measurements and hence assignments of identities of the molecules including potential PTMs. Based on the mass measurements, N-terminal pyroglutamate (pyro-E) of the heavy chain at 50668.23 Da and of the light chain at 23396.6189 Da, as well as oxidized form of the heavy chain at 50702.20 Da are readily identified in the RH4 stability sample (Figure S1). Although the intact and DTT-reduced mass values of the impurities and variants were applied to provide clues of the protein identities, many of them are not sufficient for confident identification of the impurities, since each mass value may have several sequence candidates even with a mass tolerance of 5.0 ppm. Therefore, more detailed sequence information from tandem MS (MS/MS) is required for confident sequence assignments. Charge-state Dependent HCD Middle-down MS/MS of the Light Chains and the Heavy Chains of Anti-C. diff mAb The HCD MS/MS was first investigated on the disulfide bond-reduced light chains of anti-C. diff mAb due to its smaller size. HCD MS/MS with NCE of 26% (91.4 eV), 28% (98.4 eV) and 30% (105.5 eV) were sequentially applied to light chain [M+12H]12+ ion (m/z 1953.39) to exam the fragmentation efficiency (Figures S5 – S7). NCE of 26% is sufficient to dissociate the light chain and affords the best sequence coverage of 31.6% by detection of 37 b-ions and 32 y-ions. At higher NCE of 28% and 30%, the numbers of both the b-ions and y-ions decrease, and the resulted sequence coverage drops to 25.6% and 20.5% respectively. HCD MS/MS were subsequently optimized on charge states selected for gas phase dissociation, because it has been reported that top-down MS/MS of proteins could be charge-state dependent,


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either by CID 49 or by ECD.35 Two precursor ions of the light chain, [M+26H]26+ and [M+18H]18+, which represent a high- and an intermediate-charge-state ions of the charge-state envelope, were selected individually for middle-down MS/MS. In each MS/MS experiment, the collision energy was optimized to obtain the best sequence coverage of each of the precursor ions as demonstrated previously for the [M+12H]12+ ion of the light chain. Fragmentation of [M+26H]26+ (m/z 902.05) yields 15 b-ions and 49 y-ions with a total amino acid sequence coverage of 29.8% (Figure S8). As compared with HCD MS/MS of the [M+12H]12+ ion, more Cterminal fragmentation and less N-terminal fragmentation are observed for the [M+26H]26+ precursor ion. This observation is consistent with the CID MS/MS by McLuckey in that fragmentation of higher charge-state precursor ions in a charge-state envelope of a protein generates better C-terminal sequence information of the protein.49 Furthermore, our HCD MS/MS of the intermediate-charge-state ion [M+18H]18+ (m/z 1302.58) (Figure S9) shows more fragmentation than the above two charge states. Almost all the observed product ions of [M+12H]12+ and [M+26H]26+ are identified in the MS/MS of [M+18H]18+, leading to a total identification of 32 b-ions and 57 y-ions corresponding to a 39.5% sequence coverage of the light chain. Based on this observation, additional intermediate-charge-state precursor ions were selected for further evaluation. The results show that MS/MS of [M+19H]19+ or [M+20H]20+ alone give not only overlapping but also some new product ions. Therefore, three intermediatecharge-state ions, [M+18H]18+, [M+19H]19+and [M+20H]20+, were simultaneously fragmented to get better sequence coverage. The data processed by ProSight PC identified 39 b-ions and 67 yions that represent sequence coverage of 46.0% of the light chain (Figure S11). Three additional intermediate-charge-state ions of the light chains, [M+15H]15+, [M+16H]16+and [M+17H]17+, were selected for simultaneous HCD fragmentation to show that the results are reproducible.


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Figure 2 demonstrates data processing results by Byonic showing the N- and C-terminal fragment ion assignments, light chain sequence coverage, and fragment ion assignment accuracies (see supporting information for more details of Byonic software). Using the Byonic software, light chain sequence coverage of 46.0% is identified by assignments of 36 b-ions and 65 y-ions with an estimated false peak assignment rate of approximately 1%, which is in well agreement with the 45.1% sequence coverage processed by ProSight PC (Figure S12). The Cand N-termini of the light chain, the Complementary Determining Region 1 (CDR1), as well as the CDR3 are extensively sequenced by this HCD middle-down MS/MS. These results demonstrated better efficiency than the previously reported HCD MS/MS experiments on IgG light chains (18.0%~ 39.5% sequence coverage, Table S3). In term of sequence coverage, this HCD fragmentation is slightly lower than the recently reported ~50% coverage by one LC run of ETD MS/MS experiments on the IgG1 light chains.17 As shown in Figure S13. Simultaneous fragmentation of three intermediate-charge-state ions, [M+28H]28+, [M+29]29+ and [M+30H]30+, generates 57 b-ions and 35 y-ions covering 20.3% of the heavy chain sequence. It is worth mentioning that since a m/z 5 isolation window is applied for tandem MS of each of the three charge states, only the G0F glycoform of the heavy chain was fragmented, which simplifies data interpretation. The result is comparable to the sequence coverage by ETD MS/MS on disulfide bond-reduced IgG heavy chains and similar molecular weight small proteins (see Table S3). HCD fragmentation of the heavy chain sequenced most of the CDR1 and CDR2 regions, which are very important for characterization of PTMs in these regions as the modification could potentially impact antibody binding to its target.50 Characterization of Anti-C. diff mAb Impurities by HCD Middle-down MS/MS


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To characterize mAb product-related impurities, anti-C. diff mAb and its impurities in the CEX Fractions 4 and 5 were disulfide-bond reduced by DTT prior to HCD middle-down LC/MS-MS. With an isolation window of m/z 5, the top three most abundant intermediate-charge-state ions of the charge-state envelope of an impurity molecule were generally selected for HCD dissociation. Specifically, HCD MS/MS of [M+13]13+, [M+14]14+, and [M+15]15+ charge states of Impurity 4 generates 36 b-ions and 26 y-ions as shown in Figure 3. The detection of a consecutive series of b2 to b7 ions confirms that this impurity share the same N-terminal glutamic acid (Glu1) as the heavy chain, suggesting it is a C-terminal truncated form of the heavy chain. Combining with its detected monoisptopic mass of 24273.9696 Da (Table S2), detection of y2, y3, y6, y8, y12 and y13 ions identifies the C-terminal amino acid residue of Impurity 4 as His226. Hence Impurity 4 is confirmed to be a fragment of the heavy chain with an intact N-terminus and a C-terminal His226. Based on this identification, the calculated intact molecular average mass of Impurity 4 (heavy chain Glu1–His226 and disulfide-linked with a light chain) matched the measured intact mass value 47707.54 Da (Table S1), with a mass accuracy of 13.7 ppm. Similarly, in combination with detected mass values of DTT-reduced and disulfide bond-linked intact molecules, the sequences of Impurities 1 – 3 and 5 – 7 are characterized by HCD MS/MS. The results of identified impurity sequences are shown in Table 1, and their HCD MS/MS spectra are shown in Figures S14 – S19. Other than Impurity 3 (heavy chain Trp102–Gly448) and Impurity 1 (C-terminal truncated form of the light chain), the impurities of anti-C. diff mAb are C-terminal truncations of the heavy chain while maintaining an intact N-terminus, such as truncations at Asn101 with a sequence of Glu1-Asn101 (Impurity 2) and at the hinge region amino acids with the sequences of Glu1–Cys222 (Impurity 6), Glu1–Asp223 (Impurity 7), Glu1–Lys224 (Impurity 5).


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Finally, when compared with the light chain eluted at 10.5 min (Figure S1), the light chain eluted at 11.5 min showed a mass decrease of 18 amu for all of its b-ions by HCD MS/MS, confirming the existence of the light chain with an N-terminal pyroglutamate in this peak. The N-terminal pyroglutamate is similarly identified for the heavy chain eluting at 20.4 min by detection of the characteristic “-18 amu” b-ions as compared with the heavy chain eluting at 18.6 min (Figure S2). Successful identification of these impurities by LC/MS-MS using HCD demonstrates that this approach can be applied for low-level mAb drug product-related impurity characterization in biologics development. Identification of Site-specific Oxidation of Heavy Chain Methionines by HCD Middle-down LC/MS-MS Methionine (Met) is an oxidation-sensitive amino acid in mAbs. Oxidation of Met observed in mAb stability studies, especially those in CDR3, could be of great concern since it could impact the activity of the molecule during long-term storage.50 MS of DTT-reduced RH4 stability sample of anti-C. diff mAb detects a 16 amu increase for the heavy chains eluting at 16.9 min and 17.8 min with detection levels of 4.9% and 5.5% by UV (Figure S1), which suggests they could be oxidized heavy chain. To identify the oxidation sites, HCD MS/MS experiment was performed on the oxidized heavy chain eluting at 16.9 min. Simultaneous HCD MS/MS of precursor ions at m/z 1690.6 (30+), 1748.8 (29+) and 1811.2 (28+) yielded fragmentations mostly similar to those of the apo-form heavy chain. Figure S20 is the MS/MS spectrum that shows the detected b34 – b52 fragment ions zoomed-in at the range of m/z 1200 – 1500. The m/z value of the b34 ion is identical to that of the apo-form heavy chain. However, each of the b40,


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b46, b47, b48, b50 and b52 fragment ions show a mass increase of 16 amu as compared with those of the apo-form heavy chain. The detection of the 16-amu mass increase of these b-ions confirms that Met40 is oxidized in the heavy chain eluting at 16.9 min. However, HCD MS/MS of the oxidized heavy chain eluting at 17.8 min did not generate sufficient product ions for the site-specific assignment of oxidation. Therefore, a pseudo MS3 by applying in-source CID prior to HCD MS/MS was employed. In-source CID of the heavy chain detects a mass increase of 16 amu for the b15410+ ion of the oxidized heavy chain as compared with that of the apo-form heavy chain, suggesting the oxidation site was within its N-terminal amino acid residues up to Glu154 (Figure 4). The (b154 + 16 amu)10+ ion was subsequently isolated as a precursor ion for further gas-phase dissociation. A total of 40 b-ions and 6 y-ions are identified in this pseudo MS3 mass spectrum, resulting in heavy chain sequence coverage of 27.3% (Figure 4). The b986+ ion of the oxidized heavy chain shows a 16-amu mass increase as compared with that of the apo-form heavy chain, while the b734+ ion remains identical. This result suggested that oxidation occurred on Met93. Similarly oxidation of Met430 is identified in the heavy chains that co-eluted with apo-form heavy chains at 18.6 min in the DTT-reduced CEX Fraction 4 by HCD MS/MS of precursor ions of m/z 1690.6 (30+), 1748.8 (29+) and 1811.2 (28+) simultaneously with an isolation window of m/z 5 (Figure S21). The level of oxidation of Met430 was estimated to be 2.7% by extracted ion chromatogram (XIC) peak areas of the y605+ ions of the apo- (m/z 1358.6) and the oxidized (m/z 1361.5) heavy chains, as shown in Figure S21. Furthermore, the identification of oxidation of Met430, as well as Met40 and Met93, is confirmed by a bottom-up LC/MS peptide mapping analysis with detected oxidation levels of 2.8%, 6.8% and 2.0% respectively by XIC of corresponding peptides (see Figure S22).


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Conclusions In summary, we have demonstrated the application of HCD middle-down LC/MS-MS to characterize the product-related impurities and variants presented in an RH4 stability sample of an anti-C. diff IgG1 mAb on a Thermo Q-Exactive mass spectrometer. The gas phase dissociation of large protein molecules by HCD is found to be both collision energy- and precursor ions’ charge state-dependent, as demonstrated in the HCD MS/MS of the light chain of the anti-C. diff mAb. With proper selection of the collision energy and the precursor ions, efficient fragmentation and hence good sequence coverage of both the light chains and the heavy chains are achieved. By combining the HCD middle-down sequencing information and the mass measurements of both the intact and the disulfide bond-reduced molecules, identification of sitespecific modifications including the clipping sites of the heavy chain (N101/W102, C222/D223, D223/K224, K224/T225, H226/T227) and of the light chain (G93/S94), as well as oxidation of heavy chain methionine residues (Met40, Met 93 and Met430) is achieved for the seven impurities collected from CEX separation of the RH4 stability sample of the therapeutic mAb. These results demonstrate that this online LC/MS and middle-down tandem MS by HCD is able to provide accurate structure characterization of the low-level (< 10%) product-related impurities presented in mAb drug product without the need for extra chromatographic separation and fractionation. Therefore, we expect this characterization method to facilitate biologics development in general by providing accurate characterization of low-level drug product-related impurities and variants in drug substance and drug product.

Acknowledgements


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The authors thank Dr. Xiaoyu Chen and Ms. Sarah M. Hall for CEX fractionation of anti-C. diff mAb stability sample. This work is supported by Merck Research Laboratory (MRL). ASSOCIATED CONTENT Supporting Information Available This information is available free of charge via the Internet at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y.-H. L.). Notes The authors declare no competing financial interest.


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1 2 3 Table 1. Sequences of heavy chain, light chain and 4 and DTT-reduced mass analysis. 5 6 Impurity Impurity 1 Impurity 2 Impurity 3 Impurity 4 7 8 9 Characterization 10 E1-G93 E1-N101 W102-G448 E1-H226 11Sequence (LC) (HC) (HC) (HC) 12 14 b-ions 10 b-ions 17 b-ions 36 b-ions 13Fragmentation ions by HCD top-down 21 y-ions 27 y-ions 21 y-ions 26 y-ions 14MS/MS 15 16Sequence coverage 25.8% 35.6% 11.0% 27.4% 17by HCD top-down MS/MS 18 19Theoretical mass 10024.9454 11276.4958 39420.83 3 24273.9479 20(Da)(monoisotopic) 21 22Mass accuracy 1.7 2.0 -6.8 0.9 23(ppm) 24 25 1. HC: Heavy Chain; 26 2. LC: Light Chain; 27 28 3. Average mass is for Impurity 3 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the impurities identified by HCD MS/MS

Impurity 5

Impurity 6

Impurity 7

HC1

LC2

E1-K224 (HC)

E1-C222 (HC)

E1-D223 (HC)

E1-G448

E1-C215

12 b-ions 20 y-ions

8 b-ions 11 y-ions

9 b-ions 15 y-ions

57 b-ions 35 y-ions

39 b-ions 67 y-ions

14.3%

8.6%

10.8%

20.3%

46.0%

24035.8413

23793.7194

23907.7464

50686.62

23414.5445

1.5

3.7

1.4

-4.5

1.6


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Figure 1. (A) ESI-MS mass spectrum of intact anti-C. diff mAb shown in the range of m/z 1800 3700. The insertion shows the 54+, 55+ and 56+ charge states with resolved N-glycosylation species. (B) Deconvoluted mass spectrum of anti-C. diff mAb.


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Figure 2. Byonic identification/annotation of HCD MS/MS of the light chains of anti-C. diff mAb by simultaneous fragmentation of [M+15H]15+, [M+16H]16+ and [M+17H]17+ charge-stage ions. (A) Fragment ion identification and annotation from a single scan MS/MS of m/z 1379.1 (17+), 1465.2 (16+) and 1562.8 (15+); (B) Fragment ion assignments zoomed on low m/z region; and (C) Fragment ion assignments zoomed on high m/z region.


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Figure 3. HCD MS/MS spectrum of simultaneous fragmentation of [M+13H]13+, [M+14H]14+ and [M+15H]15+charge states of DTT-reduced Impurity 4. Only major fragment ions are labeled. The sequence coverage map is shown on the top of the spectrum.


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Figure 4. (A) In-source CID MS/MS spectrum with b15410+ of the oxidized heavy chain shown in m/z 1300 - 1800; and (B) HCD MS/MS spectrum of the in-source CID generated b15410+ ion. The zoom-in figures compare b986+ (insertion on the left) and b-734+ (insertion on the right) ions from HCD MS2 of the b15410+ ion of the oxidized heavy chain (top) and the apo-form heavy chain eluting at 18.6 min (bottom), respectively. The sequence coverage map of the b15410+ is shown on the top of the spectra.


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Haberger, M., Bomans, K., Diepold, K., Hook, M., Gassner, J., Schlothauer, T., Zwick, A., Spick, C., Kepert, J. F., Hienz, B., Wiedmann, M., Beck, H., Metzger, P., Mølhøj, M., Knoblich, C., Grauschopf, U., Reusch, D., Bulau, P. mAbs 2014, 6, 327–339.


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Table of Contents


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Table of Content 47x28mm (300 x 300 DPI)


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Characterization of Drug-Product-Related Impurities and Variants of a

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