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Characterization of IgG2 Disulfide Bonds with LC/MS/MS and Post-column Online Reduction Hongji Liu, Qing Paula Lei, and Michael Washabaugh Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04368 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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Analytical Chemistry

Characterization of IgG2 Disulfide Bonds with LC/MS/MS and Post-column Online Reduction Hongji Liu, Qing Paula Lei and Michael Washabaugh Analytical Biotechnology, MedImmune, One Medimmune Way, Gaithersburg, MD 20878, USA

Corresponding author: Hongji Liu Email: [email protected] Phone: 001-301-398-2034

ACS Paragon Plus Environment


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Abstract The complication of IgG2 disulfide connections demands advances in techniques for disulfide bond determination. We have developed a new LC/ MS/MS method for improved disulfide analysis. With post-column introduction of dithiothreitol (DTT) and ammonium hydroxide, each disulfide-containing peptide eluted out of LC in an acidic mobile phase can be rapidly reduced prior to MS analysis. The reduction can be driven to near completion. The reagents are MS-friendly and the reaction occurs at no cost of separation (little is added to the post-column dead volume of the LC system). Comparing LC/MS data with and without online reduction, a direct correlation can be established between a disulfide peptide and its composing peptides using retention time. With disulfide online removal, high quality MS/MS fragmentation data can be acquired and allows for definitive determination of the disulfide peptide. This technique is especially valuable in determining the disulfide bond linkage of complicated molecules such as the hinge-containing disulfide peptides produced from IgG2 disulfide isoforms. Due to over/under enzymatic cleavages, multiple hinge-containing disulfide peptides are produced from each isoform. Twenty-two hinge-containing disulfide peptides in total have been confidently identified with this technique. Without the method, successful identification to many of these peptides would have become extremely difficult.

Introduction The correct formation of disulfide bonds within a protein is critical for its desired function and stability1. Inappropriate disulfide connections might lead the protein to lose its bioactivity and to aggregate, generating potency and safety issues for its therapeutic applications. For this reason, the regulatory agencies like the Food and Drug Administration and the International Conference Harmonization for new drug and biosimilar applications require disulfide bond characterization as part of structural characterization2,3. The characterization is necessary for recombinant proteins as, even though their disulfide bonds are usually formed as predicted during expression or refolding, the formation of unpredictable disulfide bonds, or disulfide scrambling, can still occur. Furthermore, with more disulfide engineering techniques being applied, where novel, and therefore, non-native disulfide bonds are introduced4-6, the chance of disulfide scrambling becomes much greater. The most commonly used technique to determine the disulfide bond connections within a protein is to first enzymatically digest the protein, reduce a portion of the digest, and then run the reduced in parallel with the non-reduced portions using LC/MS7-10. Any difference in the chromatographic profiles of the two portions may indicate the presence of a disulfide connected or disconnected peptide. Ideally, a disulfide peptide and its structure should be directly characterized by LC/MS/MS. In reality, however, direct analysis is difficult to achieve due to the presence of disulfide bond(s) in each peptide, which may severely affect the quality of the spectra produced with collision induced dissociation (CID) 11-13, the most commonly used data for peptide identification. Recently, several LC/MS techniques have been successfully applied for direct disulfide elucidation, which include those with CID under a negative mode14,15 , with electron-transfer dissociation (ETD) 16,17, and with the classic N-terminal sequencing


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chemistry 18,19. Before these new techniques are widely applied, however, the identity of a disulfide peptide may still be confirmed indirectly, by measuring its mass from the non-reduced map, by detecting its disappearance after reduction, and by finding its components (i.e. reduced peptides) in the reduced map. Due to the presence of multiple disulfide peptides in the same digest, no direct relationship can be established between any disulfide peptide and its components. As a result, the composition of a disulfide connected peptide, which could contain multiple composing peptides, can only be conjectured. Sometimes, even a conjectured composition can be difficult to obtain due to many factors, such as unexpected changes in protein structure (e.g. oxidation, deamidation, fragmentation, disulfide scrambling), or some unexpected behaviors of the enzyme used (e.g. missed or non-specific digestion). Suggesting a possible composition for such an unknown disulfide peptide can be time consuming, and can sometimes fail. Even if a possible composition is suggested, it is still difficult to verify. The more complicated the unknown disulfide peptide, the more difficult its composition can be conjectured and verified. IgG 2 is a subclass of antibody composed of multiple complex disulfide structures with three disulfide isoforms (A, A/B and B) for each protein20-24. The disulfide bond characterization for such a protein is more difficult than others, due to not only the large number but also the structural complexity of the disulfide peptides to be identified. For example, a Lys-C disulfide peptide produced from Isoform B contains 8 peptide chains connected by 8 disulfide bonds, with a molecular weight (MW) over 25 kDa. By conjecture, it would be very difficult to figure out the composition of such a complex structure. In fact, this peptide was first identified by collecting its LC peak, reducing the collected peptide or its digestion products, and then loading its components back to the LC/MS system20,23. This offline reduction approach might be practical for a high-abundance peptide, but may become more difficult to carry out for less abundant ones. When working on disulfide characterization for an IgG2 drug candidate, we observed many disulfide peptides that were difficult for us to conjecture. Most of them had very low abundances with high MWs. Identifying the composition for each of these disulfide peptides with confidence led us to develop this new online reduction method. Disulfide online reduction has previously been used by others with either a chemical25,26 or electrochemical mechanism12,13,27-31 32 33. However, except for a few exceptions 26 32 33, all the online reductions were applied without LC separation. In the chemical approach, tris(2carboxyethyl)phosphine (TCEP) was used as the reduction reagent25,26. TCEP can reduce disulfide bonds in an acidic environment, and is therefore compatible with the disulfide LC/MS/MS mapping which is usually conducted in an acidic mobile phase. However, under the low pH of 2-3 in the mobile phase, the reduction rate seems very low resulting a great portion of each disulfide peptide remaining unreduced 25,26 .Furthermore, TCEP seems incompatible with MS since it is positively charged, non-volatile and usually chloride-containing, even though its direct infusion to MS has been tried26. The electrochemical reduction approach is MS compatible, but requires a reaction cell which inevitably increases the dead volume of the LC system and hence affects separation, especially at a low flow rate as preferred by this technique to increase its reduction efficiency 13 32 33.. Ion suppression was also observed 32. The reduction rates reported or indicated from these applications vary greatly, but mostly not close to 100%. Notably, in one application, oxidation has even been observed during reduction31.



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In the present post-column online reduction method, DTT is employed as the reduction reagent. DTT has been widely utilized for disulfide reduction, but has never been applied online. The reduction requires a basic environment, in contrast to the acidic one commonly used for disulfide mapping. The reduction is usually slow, requiring 20-30 minutes to complete, and therefore incompatible with online reaction which usually requires the reaction to complete in a few seconds. However, these issues can be readily solved by post-column introduction of ammonium hydroxide (NH4OH). At pH≥10, most disulfide peptides can be fully or mostly reduced. No DTT or any other adducts have been observed. The resulting reaction is so rapid that no additional reaction cell is needed, and therefore LC separation can be fully unaffected.

Experimental Section Materials Digests of an IgG2 drug candidate (Antibody X) produced in-house were prepared. The antibody consisted of two κ light chains and two heavy chains in each molecule, Lys-C was produced by Wako Chemicals (Richmond, VA, USA). Smoky NH4OH and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2M NH4OH was produced by Ricca (Arlington, TX, USA). DTT (Noweigh format) was a product of Thermo Scientific (Rockford, IL, USA). Bis(2-Mercaptoethylsulfone) or BMS, HPLC grade water and acetonitrile were all produced by EMD Millipore (Billerica, MA, USA). LC/MS/MS conditions Online reduction was performed post-column within an LC/MS system (Waters, Milford, MA, USA). The system consisted of an Acquity UPLC unit and a Q-Tof Premier or LCT Premier mass spectrometer unit. A Waters Acquity BEH or CSH C18 (1.7 µm, 2.1 x 150 mm) column (thermostated at 60 °C) was connected for separation (column change introduced some different separations rather than different mass recoveries). The mobile phase used consisted of two eluents: Eluents A and B contained 0.1% TFA in water and acetonitrile, respectively. The gradient applied was 0% to 45% B for 46 min at a flow rate of 0.2 mL/min. The antibody was digested using Endoproteinase Lys-C with a procedure which was essentially the same as previously described20. The digests were concentrated to increase the amounts loaded into LC/MS. An injection of digest equivalent to approximately 5 µg intact protein was made each time. For electrospray ionization, the source and desolvation temperatures were maintained at 130°C and 500 °C, respectively, the desolvation gas flow was set at 600 liters/hour, and the capillary and the sampling cone voltages were adjusted to 3.1 kV and 40 V, respectively. The MS data were acquired in a mass range of 200-3500 Da. As needed, MS/MS was acquired with an MSE mode using the Q-Tof Premier unit. LC/MS conditions were controlled and data collected with Waters MassLynx software. Waters MaxEnt1 software was employed for spectrum deconvolution for high-MW peptides whose monoisotopic masses were difficult to measure. From the deconvolution, averaged masses data were obtained. Online reduction


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The post-column online reduction configuration is shown in Figure 1. DTT was introduced by infusing a 0.1 M DTT solution with a syringe pump. The pH adjustment to the mobile phase was made possible by pumping an NH4OH solution with another syringe pump. The syringes and syringe pumps were products of Hamilton (Reno, NV, USA) and KD Scientific (Holliston, MA, USA), respectively. The mobile phase, the DTT and the NH4OH fluids were mixed at a cross (0.02” through holes, 0.72 µL total volume) produced by Upchurch (Oak Harbor, WA, USA). Unless otherwise indicated, the cross was placed between the column and the UV detector (flow cell: 0.5 µL). The size of the tubing connecting to the UV unit and the MS unit was 0.007” (internal diameter) x ~60 cm (length). A diversion valve (Waters), which had a volume of 2.2 µL/hole and 0.4 µL/groove, was placed after the UV detector (not shown in Figure 1). 0.1 M DTT

HPLC

UV

MS

Column NH4OH

Figure 1: Configuration of post-column online reduction

Results and Discussion Online reduction of disulfide peptide H23-H27 H23-H27 is a Lys-C disulfide peptide of Antibody X composed of the 23th (sequence: NQVSLTCLVK) and 27th (sequence: SRWQQGNVFSCSVMHEALHNHYTQK) peptides of its heavy chains. The two composing peptides are linked with a disulfide bond connecting two cysteine residues, one from each peptide. Figure 2 demonstrates online reduction of its disulfide bond with DTT. A non-reduced Lys-C digest of Antibody X was injected, and, from the obtained total ion current (TIC) chromatogram, the most intensive masses of H23-H27 and its reduction products were each extracted. In panel (a), H23-H27 eluted at 30.4 minutes in its non-reduced form when no DTT and NH4OH was introduced. Here the online reduction setup was removed to completely eliminate the effect of the setup on separation. In Panel (b), with the introduction of DTT and NH4OH (0.1M DTT at 10 µL/min and 2M NH4OH at 7.2 µL/min), the peptide eluted at the same location, but 95% of it was reduced, with its reduction products, the peptides H23 and H27 being both observed. In Panel (c) the reaction was performed with the same DTT fluid as in Panel (b), but the NH4OH flow rate was increased to 12 µL/min. This time, with the increase in pH for reaction, almost all the disulfide peptide molecules were reduced. The peak widths and retention times of H23 and H27 were essentially the same as H23-H27, indicating unaffected chromatographic performance of the system by the post-column reaction configuration. Note that only the reduced peptides eluted at 30.4 min can possibly belong to H23-H27, and any other peptide (such as the one eluting at 28.9 min) are certainly not part of H23-H27. In fact, the reduced peptide shown at 28.9 min (marked with *) was H23 produced from a disulfide peptide composed of H23 and (o)H27, the



oxidized H27. The mass of H23-(o)H27 is shifted out of the mass extraction window set up for H23-H27, and therefore the H23-(o)H27 peak is not shown at 28.9 min even with the online reduction being taken off.

%

%

%

C_HJL20140423_002 F1 C_HJL20140423_002 F1 F1 C_HJL20140423_002 996.191_998.67 1104.512_1108.12 H23-H27 1022.621_1025.86 1.95e4 100 3.67e4 100 2.94e4 100

(a) 0

%

%

(b)

%

0 0 30.00 35.00 30.00 35.00 30.00 35.00 C_HJL20140423_006 F1 C_HJL20140423_006 F1 F1 C_HJL20140423_006 1022.84_1025.37 996.281_999.39 H23 1104.441_1108.36 H27 1.95e4 100 3.67e4 100 2.94e4 100

*

H23-H27 0

%

(c)

%

0 0 30.00 35.00 30.00 35.00 30.00 35.00 C_HJL20140423_008 F1 C_HJL20140423_008 F1 F1 C_HJL20140423_008 1023.371_1025.42 996.262_999.48 1104.481_1108.37 H27 H23 1.95e4 100 3.67e4 100 2.94e4 100

%

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* 0

Time 30.00

35.00

0

Time 30.00

35.00

0

Time 30.00

35.00

Figure 2: Online reduction of H23-H27 (a): no online reduction; (b): online reduction with 0.1M DTT at 10 µL/min and 2M NH4OH at 7.2 µL/min; (c): online reduction with 0.1M DTT at 10 µL/min and 2M NH4OH at 12 µL/min.

Figure 3 compares the raw MS spectra acquired at a location where H23-H27 or its reducing products eluted. As previously demonstrated (Figure 2c), H23-H27 was almost undetectable after online reduction (the arrow in Figure 3b points to the sole observed mass). The spectrum acquired with online reduction was quite clean, with no ammonium, DTT or TFA adducts being detected. The two unknown ions of 359.14 Da and 513.18 Da (both singly charged, each marked with an asterisk in Figure 3b) were observed with online reduction, but their masses and intensities were relatively low and would not affect peptide detection. The source of these unknown ions was not identified, but using purer DTT and NH4OH did result in some reduction in their magnitudes. Note that there were some significant sodium adducts being observed in Figure 3b. However, these adducts were not produced with online reduction,


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%

%

1 2 3 but related to charge state changes. For example, the singly charged H23 had the highest adduct 4 formation, as indicated by a significant 1126.83 Da peak. The adduct formation was significantly lowered 5 6 for the doubly charged H23, indicating a downward trend for the adduction formation at a higher charge 7 state. This can explain why minimal adduct was observed for the highly charged (≥+3) H23-H27 (the only 8 observed adduct had a mass of 1371.95 Da, formed with the triply charged H23-H27). This explanation 9 10 was supported with a singly charged non-disulfide peptide. This peptide exhibited similar degrees of 11 sodium adduct formation under conditions with and without online reduction (data not shown). 12 13 14 15 +4 C_HJL20140423_002 1529 (30.248) Cm (1523:1535) 1: TOF MS ES+ 16 C_HJL20140423_002 1529 (30.248) Cm (1523:1535) 3.49e3 1023.72 +3 100 17 a 1023.47 1023.97 1364.64 18 1364.30 1364.94 1024.23 19 1365.30 20 1023.24 1363.98 21 1024.46 1365.62 +5 +6 22 1024.71 819.17 1365.98 1371.95 682.64 818.77 819.57 23 0 24 25 C_HJL20140423_008 1527 (30.225) Cm (1523:1535) 1: TOF MS ES+ H23(+1) C_HJL20140423_008 1527 (30.225) Cm (1523:1535) 1.24e4 1104.81 26 100 H23(+2) b 27 H27(+3) 997.01 28 552.92 1105.85 29 996.69 997.34 1126.83 553.43 997.70 30 1127.85 31 998.05 * 553.94 H27(+4) * 359.14 513.18 1149.85 32 H27(+2) 998.37 564.44 748.02 33 0 m/z 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 34 35 36 37 Figure 3: MS spectra of H23-H27 acquired with and without online reduction 38 39 40 (a): without online reduction, (b): with online reduction (0.1M DTT at 10 µL/min and 2M NH4OH at 12 41 µL/min) 42 43 After online reduction, the composing peptides of H23-H27 were readily identified with MS/MS (Figure 44 4) 45 46 47 48 49 50 51 52 53 54 55 56 Figure 4: MS/MS spectra composing peptides of H23-H27 57 58 59 60 ACS Paragon Plus Environment


(a): H23 and (b): H27

Effect of pH on reduction of H23-H27 Figure 2 has shown the effect of mobile phase pH (after adjusted with NH4OH) on the reduction of H23H27. This effect can be further described in Figure 5 where the detected MS signals for the disulfide peptide and its reduction products are plotted against reaction pH. Note that both the MS signals and the reaction pH were measured indirectly. In absence of a pH probe equipped in the LC/MS system, it was impossible to monitor the pH value online. Instead, each value was estimated offline with a pH meter. By measuring the pH values of mixtures of 0.1% TFA (in water) and NH4OH solutions at various volumetric ratios, a titration curve was plotted (not shown), from which the adjusted pH values of the mobile phase were estimated (without considering the effect of organic content on pH). The MS signals of the reduced and non-reduced peptides were normalized against a non-disulfide peptide (retention time: 29.4 min) eluting next to them. The normalization was performed to minimize the effect of mobile phase pH change (after basification) on the MS signals of the peptides of interest. As indicated from the plots in Figure 5, the online reduction of H23-H27 was greatly affected by the reaction pH. Unsurprisingly, little H23-H27 was found reduced at pH8.2 where an offline reduction is usually conducted. At such a pH, offline reaction usually takes 20-30 minutes, at least 100 times longer than the ~10 seconds of reaction time as provided by the current configuration of online reduction. However, with the increase of reduction pH, more disulfide molecules were reduced online. After the pH reached 9.2 (the value of DTT’s pKa), ~50% of H23-H27 molecules were reduced. At pH 10.3, the highest pH point tested, 99% of H23-H27 molecules were converted to their composing peptides.

1.5 H27 H23

Relative peak area

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0

H23-H27

8

Reduction pH

10

Figure 5: Effect of pH on reduction of H23-H27

Effects of post-column volume and DTT flow rate on H23-H27 online reduction


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The online reduction of H23-H27 occurs after it is mixed with DTT at the cross. Unless the reduction happens instantly and does not require any time for reaction, the reduction should be affected by the time of reaction, which is the duration of time after the reaction components are mixed but before the peptide is detected by MS. At a constant HPLC flow rate (in this study, we maintained the flow rate at 0.2 mL/min), the time of reaction relies on the post-column volume. Figure 6 shows the effect of the post-column volume on the reduction of H23-H27. Note that, a newly prepared digest was employed for this experiment. As a result of a less degree of post-digestion oxidation, the concentration of H23-H27 in the current digest was increased, and therefore, after full reduction, the observed peak areas of H23 and H27 were somewhat higher than those observed in other experiments of this study. Due to the expected high reaction speed at pH10.3, which would likely lead to full reduction even at very short reaction time and make little diffidence in the reduction ratios at various post-column volumes, the experiment was performed at pH10, a less optimized reaction condition (2M NH4OH flow rate: 10 µL/min, DTT flow rate: 10 µL/min). Based on the relative peak response of H23, 91% of H23-H27 was reduced at a 13 µL post-column volume (equivalent to 3.5 seconds in reaction time), a volume less than that given by normally configured LC/UV/MS system. However, driving the reduction to completion would need approximately 50 µL (reaction time: 13.6 seconds), a volume greater than the post-column volume intrinsically provided by the LC/MS system used (~30 µL or 8.2 seconds for) . For complete reduction at pH10, one could choose to deliberately increase the reaction time by, for example, adding a piece of tubing. However, since doing so will deteriorate the achieved separation, increasing reaction pH is more desirable. 2 H27

Relative peak area

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H23

H23-H27

0 0

Post-column volume (µl)

60

Figure 6: Effect of post-column volume on reduction of H23-H27 (pH 10) In this experiment the configuration shown in Figure 1 was modified so that, except for the ESI source, all the post-column components were temperately moved upstream. Between the cross and the ESI housing a piece of tubing was placed. The post-column volume in the system was represented by the tubing volume.

The effect of DTT flow rate on online reduction of H23-H27 is shown in Figure 7. Complete reduction was achieved when the 0.1 M DTT was pumped at 10 µL/min. Almost complete reduction was still observed at a 5 µL/min flow rate, but incomplete reduction became significant when the flow rate was



reduced to 2 µL/min. This experiment was carried out at pH10.3, the most basic condition tested in this study. It can be speculated that the effect of DTT flow rate on reduction will be more significant at a lower pH. Therefore, it is necessary to pump the 0.1M DTT at 5 µL/min or higher for a more complete reduction. Except for this experiment, a 10 µL/min flow rate of 0.1M DTT was employed for the whole study.

1.4 H27 H23

Relative peak area

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H23-H27

0 1

0.1 M DTT flow rate (µl/min)

11

Figure 7: Effect of flow rate of 0.1M DTT on reduction of H23-H27 Reaction pH: 10.3

No effect of DDT concentration on online reduction was investigated as it was expected that the effect would be about the same as that of the DTT flow rate. It seems necessary to keep the concentration at 0.1 M or higher for the reduction. Even though one could choose to use a less concentrative DTT by introducing it at a higher flow rate, pumping a much more volume of liquid into the system is undesired as doing so will burden ionization, reduce the MS sensitivity and shorten the reaction time. And also, a much bigger syringe has to be in place to keep online reduction in function during the whole gradient. The same consideration should be given before a more concentrative NH4OH is replaced with a less concentrative one.

Online reduction of other disulfide peptides of Antibody X Nine disulfide peptides are expected in a Lys-C digest of Antibody X (excluding those with over/under cleavages and those produced due to post-translational modification such as oxidation or deamidation). Table 1 lists six of these peptides and their compositions. The other three peptides, all hinge-containing, will be discussed in a later section. All these nine peptides were effectively reduced with the current online configuration. Among these peptides, seven of them behaved similarly to H23-H27, with reduction ratios close to 100%. These included the peptides with multiple disulfide bonds, and the three hinge-containing ones with MWs up to 25 kDa. However, the reductions of two relatively simple peptides were slower, with that of H13-H17 being the slowest. Connecting H13 to H17, a peptide of 2


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amino acid residues, H13-H17 was never seen reduced completely, with the highest reduction rate being about 65%. As a result, H13-H17 co-existed with H13 and H17 (note that online reduction enabled the detection of the hydrophilic peptide H17 which would be impossible if with offline reduction). It is unclear why this peptide was so difficult to reduce, but obviously it is not due to structure complexity. In efforts to increase the online reduction rate of this peptide, BMS, a stronger reduction reagent than DTT, was tried but no improvement was observed (data not shown). Table 1: Six expected disulfide peptides of Antibody X and their compositions disulfide peptide

Composing peptide

L1L2-L4 L7-L13

L14-H6-H7H8

H1-H5 H13-H17 H23-H27

Residue number

Sequence

Monoisotopic mass (Da)

L1L2

1-42

DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGK

4645.25

L4

46-103

LLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTK

6238.97

L7

127-145

SGTASVVCLLNNFYPREAK

2068.04

L13

191-207

VYACEVTHQGLSSPVTK

1817.90

L14

208-214

SFNRGEC

811.33

H6

130-155

GPSVFPLAPCSRSTSESTAALGCLVK

2577.29

H7H8

156-218

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK

6705.23

H1

1-43

J*VQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK

4517.25

H5

77-129

NTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTK

6012.81

H13

253-292

DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK

4556.16

H17

325-326

CK

249.11

H23

365-374

NQVSLTCLVK

1103.60

H27

419-443

SRWQQGNVFSCSVMHEALHNHYTQK

2986.37

Disulfide linkage C23-C88 C134-C194

C214-C139 C152-C208

C22-C96 C265-C325 C371-C429

J*: pyroglutamate

Partial reduction complicates MS spectra and reduces MS intensities of the reduced peptides thereby lowering the MS data interpretation efficiency. The most time-consuming part of disulfide data analysis is on determining whether or not a peptide contains any disulfide. If the peptide disappears after online reduction, it can be almost 100% certain to be disulfide containing. But if it is still present with a reduced intensity after online reduction, it will take time to figure out whether the decrease in intensity is due to partial reduction, or is caused by other factors. Another benefit of complete reduction is the simplification of the resulting MS spectrum. Note that partial reduction can be advantageous over complete reduction in that only one injection of each digest is required for analysis. Since data for both the reduced and non-reduced forms of each peptide are required, two injections of each digest, one with one without online reduction, are needed if the reduction is driven to completion. Overall, however, complete or near complete reduction is much more desirable for disulfide data interpretation.

Other factors affecting online reduction The overall LC/MS performance is not affected by online reduction. As previously demonstrated34, peptides in a basic solution can still be detected with a positive mode, and some of the peak responses are even higher than without being basified. Due to the introduction of singly charged ions of 359.14 Da and 513.18 Da (Figure 3), the TIC baseline of online reduction was somewhat raised with online



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reduction (chromatogram not shown), but this did not affect MS data interpretation. Antibody X has two Asn residues, each of which is connected with a glycine residue on its C-terminal side. These Asn residues are more susceptible to deamidation than any other residues of the molecules. However, no change has been observed in the isotopic distribution for each of the peptides carrying these Asn residues (profiles not shown), indicating no noticeable deamidation during online reduction. The infused DTT is most likely in a liquid form in the ESI source (DTT’s melting point is 42-43 °C and its boiling point is 125-130 °C at 2 mmHg35). Little DTT smell was noted in our well-ventilated lab.

Characterization of hinge-containing disulfide peptides of Antibody X Among the three disulfide isoforms of each IgG2, the biggest difference in disulfide connections is located in the hinge region20. Under perfect digestion with Lys-C (with KP prohibition), only one hingecontaining characteristic disulfide peptide is produced from each isoform. The structure of the expected characteristic peptide for each isoform of Antibody X is shown in the left panel of Figure 8: for Isoform A, the peptide (PA1) consists of two hinge-containing peptide chains only (both H11H12) while for Isoforms A/B and B, their characteristic peptides (PAB1 and PB1) are composed of not only the hinge-containing but also other peptides. It is worth noting that, since all the involved peptides are located in the constant region, the composition of PA1 should be the same for all the IgG2 antibodies, and PAB1 and PB1 should be the same for all IgG2 antibodies when containing the same type of κ light chains. Even for those IgG2 antibodies containing λlight chains, their PAB1 and PB1 peptides are essentially the same as shown here (the sequence of L14 is replaced with TVAPTECS). Therefore, a thorough characterization for the hinge-containing peptides of Antibody X can benefit the characterization of not only Antibody X but also any other IgG2 antibodies. In theory, only three hinge-containing peptides should be present in a Lys-C digest of Antibody X. However, a much bigger number of peptides were observed in the digest. We sought to identify these peptides and determine if they were produced from the existing isoforms or corresponded to any new types of disulfide isoforms. The current method allowed us to carry out high-confidence identification for each of these hinge-containing peptides. It turned out that multiple hinge-containing peptides were produced from each disulfide isoform simply due to under and/or over digestion. Including the expected peptide, up to three, seven and twelve hinge-containing peptides have been observed for Isoforms A, A/B, and B, respectively (mostly co-present in each individual digest). Assuming the combined abundance of all these peptides being 100%, the abundance of each of peptides was mostly in the range of 1- 10%. PB5, the most abundant hinge-containing peptide, had an abundance level of 15%. The table


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on the right panel of Figure 8 describes the structures of the other detected characteristic peptides. PA1:

H11H12:

CCVECPPCPAPPVAGPSVFLFPPKPK

H11H12:

CCVECPPCPAPPVAGPSVFLFPPKPK

H7: DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK

PAB1:

H6: GPSVFPLAPCSRSTSESTAALGCLVK L14: SFNRGEC

H11H12: CCVECPPCPAPPVAGPSVFLFPPKPK H11H12: CCVECPPCPAPPVAGPSVFLFPPKPK

H7: DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK H6: GPSVFPLAPCSRSTSESTAALGCLVK

PB1:

L14: SFNRGEC

H11H12: CCVECPPCPAPPVAGPSVFLFPPKPK

L14: SFNRGEC

H11H12: CCVECPPCPAPPVAGPSVFLFPPKPK

Isoform A Name Structure PA1 PA2 PA1 -1 PK PA3 PA1 -2 PK

Mass* 5350.6 5125.4 4900.3

Isoform A/B Name Structure PAB1 PAB2 PAB1 -1 PK PAB3 PAB1 -2 PK PAB4 PAB1+1TVERK PAB5 PAB1+2TVERK PAB6 PAB1-1PK+TVERK PAB7 PAB1-1PK+2TVERK

*: Mass: mono isotopic mass; MW: average mass **: missing cleavage between H5 and H6 (sequence of H5: NTLYLQMNSLRAEDTAVYYCARDPRGA TLYYYYYGMDVWGQGTTVTVSSASTK)

MW* 15450.8 15225.5 15000.2 16064.5 16678.2 15839.2 16452.9

Name PB1 PB2 PB3 PB4 PB5 PB6 PB7 PB8 PB9 PB10 PB11 PB12

Isoform B Structure PB1 -1 PK PB1 -2 PK PB1 +1 TVERK PB1 +2 TVERK PB1 -1 PK+1TVERK PB1 -1 PK +2TVERK PB1 -2 PK +1 TVERK PB1 -2 PK +2 TVERK PB1-1 PK-1 PSNTK PB4+(H1-H5)** PB5+(H1-H5)**

MW* 25547.0 25321.7 25096.4 26160.7 26774.5 25935.4 26549.1 25710.1 26323.8 24794.1 36677.5 37291.3

H6: GPSVFPLAPCSRSTSESTAALGCLVK H7: DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK

Figure 8: Structures and theoretical masses of hinge-containing disulfide peptides detected in digests of Antibody X The structures of the expected peptides are shown on the left. Not all peptides were necessarily present in the each digest. Expectation was based on the structure of each isoform, and on the predicted Lys-C cleavage performance (with KP prohibition). The symbol of + or – indicates addition or removal of a section of sequence to an expected peptide. From the sequence shown on the left, one can readily figure out where a removal happens. For the sequence of TVERK, it adds to the N-terminus of the hingecontaining peptide.

As an example, the identity of PB4 was determined with online reduction based on the MS data shown in Figure 8. Without online reduction, a disulfide peptide with a measured MW of 26162 Da was detected. With online reduction, the disulfide peptide disappeared and was replaced with several other smaller peptides at the same chromatographic location. Since these peptides contained no disulfide bond, they were readily characterized with MS/MS (spectra not shown) as L14, H6, H7, H11 and H10H11, a product of missing cleavage between H10 (sequence: TVERK) and H11. This disulfide peptide was therefore assigned as PB4, a disulfide peptide with a theoretical MW of 26161 Da. Note that in Figure 8 some of the identified peptides contain up to 10 peptides with MWs over 37 kDa. Without online reduction, it would have been very difficult to identify these complicated peptides. Also note that over digestion (as shown by the cleavage at KP) was observed, indicating that the production of multiple hinge-containing peptides from each isoform was not simply due to insufficient digestion.



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Figure 8: Identification of PB4 with online reduction (a) without online reduction, (b) with online reduction

It is worth noting that, like some other LC/MS techniques, this online reduction method determines the disulfide connection within a peptide by identifying its composing peptides. If the disulfide peptide carries only one disulfide bond, the characterization for this disulfide can be 100% accurate. However, if the peptide contains two or more disulfide bonds, no detailed disulfide bond connections can be further revealed by the online reduction method itself. To further characterize the disulfide bonds in such a peptide, one needs to either use a technique (e.g. negative mode CID or ETD) capable of directly characterizing it, or find a method to either chemically18,19 or enzymatically break it down. We used trypsin to further digest a few multi-disulfide containing peptides of Antibody X (without fraction collection) and successfully confirmed their expected structures. However, due to the absence of a suitable enzyme to separate each of the four disulfide bonds located in the hinge region, no definitive characterization to these disulfide bonds was made possible. In fact, the hinge disulfides shown in Figure 8 were drawn based on the demonstrated structures of the disulfide isoforms for other IgG 2 proteins20. Confirming the disulfide connection details within the hinge region is beyond the ability of the current method.

Conclusions We have successfully demonstrated a new and effective post-column online reduction method for characterizing disulfide bonds of IgG2 antibodies. The method has been employed not only for Antibody X but also for other IgG and non-IgG proteins. Many disulfide peptides previously unknown to us have been successfully identified. No significant application or instrumental issues have been observed. In the future, this method can be used for top-down characterization of disulfide-containing proteins.


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However, it may not be applicable to protein preparations that require an acidic environment, such as those used for hydrogen/deuterium exchange analysis.

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(25) Tzanavaras, P. D.; Mitani, C.; Anthemidis, A.; Themelis, D. G. Talanta 2012, 96, 21-25. (26) Li, X.; Wang, F.; Xu, W.; May, K.; Richardson, D.; Liu, H. Analytical biochemistry 2013, 436, 93-100. (27) Li, J.; Dewald, H. D.; Chen, H. Analytical chemistry 2009, 81, 9716-9722. (28) Zhang, Y.; Dewald, H. D.; Chen, H. Journal of proteome research 2011, 10, 1293-1304. (29) Zhang, Y.; Cui, W.; Zhang, H.; Dewald, H. D.; Chen, H. Analytical chemistry 2012, 84, 3838-3842. (30) Nicolardi, S.; Deelder, A. M.; Palmblad, M.; van der Burgt, Y. E. Analytical chemistry 2014, 86, 53765382. (31) Trabjerg, E.; Jakobsen, R. U.; Mysling, S.; Christensen, S.; Jorgensen, T. J.; Rand, K. D. Analytical chemistry 2015, 87, 8880-8888. (32) Cramer, C. N.; Haselmann, K. F.; Olsen, J. V.; Nielsen, P. K. Analytical chemistry 2016, 88, 1585-1592. (33) Switzar, L.; Nicolardi, S.; Rutten, J. W.; Oberstein, S. A.; Aartsma-Rus, A.; van der Burgt, Y. E. Journal of the American Society for Mass Spectrometry 2016, 27, 50-58. (34) Liu, H.; Xu, B.; Ray, M. K.; Shahrokh, Z. Journal of chromatography. A 2008, 1210, 76-83. (35) The Merck Index, 13th Edition ed.; Merck & Co., Inc: Whitehouse Station, NJ, 2001.

1.5

H23-H27

DTT online Reduction

Relative peak area

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H23 + H27 H27 H23

H23-H27

0 8

Reduction pH

10.5

Abstract graphic


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MS and

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