Native Intact Mass Determination of Antibodies Conjugated with

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Native Intact Mass Determination of Antibodies Conjugated with Monomethyl Auristatin E and F at Interchain Cysteine Residues John F. Valliere-Douglass,* William A. McFee, and Oscar Salas-Solano Seattle Genetics, 21823 30th Drive South East, Bothell, Washington 98021, United States ABSTRACT: We present here a method for the rapid determination of the intact mass of noncovalently associated antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. By analyzing the antibody-drug conjugate (ADC) using native desalting conditions, we maintain the intact bivalent structure of the ADC, which ordinarily would decompose as a consequence of denaturing chromatographic conditions typically used for liquid chromatographic−mass spectrometric (LC−MS) analysis. The mass of the desalted ADC is subsequently determined using standard desolvation and ionization conditions. Methods presented previously in the literature for analyzing interchain cysteinyl-linked ADCs are either not amenable to online mass spectrometry or result in the denaturing dissociation of conjugated HC and LC during chromatographic separation and subsequent mass measurement. We have avoided this outcome with our method and have successfully and routinely obtained intact mass measurement of IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at interchain cysteine residues. Our results thus represent the first reported direct measurement of the intact mass of an ADC conjugated at interchain cysteine residues.

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have been described previously are examples of this class of molecules.7−9 This ADC class is distinct from the other types described above because the molecule is comprised of a composite of covalent and noncovalently associated LC and HC subdomains due to the presence of drugs at the interchain cysteine residues (Figure 1). The acquisition of intact mass spectral data for ADCs, which are conjugated to lysine residues or to free cysteine residues that have been engineered into non-CDR is relatively straightforward because the interchain disulfide bonds of antibodies have not been disturbed. Methods used for analyzing these constructs have been well documented.4,6,10 However, ADCs with interchain cysteine linked drugs have not previously been analyzable by intact mass methods due to the harsh solvent conditions of typical LCMS methods which disrupt the noncovalent associations between ADC subdomains. Furthermore, chromatographic methods that would both separate individual species and preserve the structure of the ADC, such as hydrophobic interaction chromatography (HIC), are not amenable to online MS detection because the separation conditions call for the use of high amounts of nonvolatile salts in the mobile phase. Additionally, HIC can separate protein

ntibody-drug conjugates (ADCs) are an increasingly important modality for treating several types of cancer. The impact of ADCs in this field is due to the exquisite specificity of antibodies which deliver the conjugated cytotoxic agent to tumor cells preferentially, thus reducing the systemic toxicity that is associated with traditional chemotherapeutic treatments.1−3 ADCs are differentiable on the basis of the drug, linker, and also the amino acid residue of attachment on the antibody. There are two main strategies for covalently linking the drug to the antibody: attachment to the epsilon amino group on lysine or the side chain thiol of cysteine. The analytical approaches to characterizing lysine conjugates have been described previously for the maytansinoid conjugate huC242DM44 and trastuzumab-DM1.5 In the case of cysteinyl-linked ADCs, there are two distinct categories. The anti-MUC16 THIOMAB-drug conjugate falls in the first category where the primary sequence of the antibody is mutated by introducing unpaired cysteine residues into the non-CDR of HC where they will not form inter- or intrachain disulfides.6 Only the unpaired cysteine residues in these engineered constructs are linked to the drug in the subsequent conjugation reactions. An alternate way of generating cysteine-linked ADCs involves partially reducing the antibody interchain disulfides prior to conjugation, which results in molecules that have a distribution of drug loading from zero to eight drugs incorporated per antibody. IgG1 vcMMAE and mcMMAF conjugates that © 2012 American Chemical Society

Received: December 16, 2011 Accepted: February 14, 2012 Published: February 15, 2012 2843

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ammonium acetate from 50 to 400 mM in 50 mM increments and varying the pH from 4 to 7 in 1 pH unit increments. In the optimized method, the column was equilibrated in 200 mM ammonium acetate, pH 7 for nondenaturing mass analysis. For denaturing mass analysis, the column was equilibrated in 30% acetonitrile and 0.2% formic acid. In contrast to the conditions described by Brady et al.,22 we did not use an organic makeup solvent and elected instead to add acetonitrile to the mobile phase and double the concentration of formic acid. The flow rate was maintained at 0.1 mL/min during the run, and the mAb or ADC was typically eluted between 3.5 and 4.5 min. The flow and buffer composition was maintained following elution of the mAb or ADC, and the total cycle time was 10 min per run. Mass Spectrometry. Mass spectral data for mAbs and ADCs was acquired on an Agilent 6510 QTOF (Agilent, Santa Clara, CA) in positive electrospray ionization (ESI) mode in the range 1000−8000 m/z. The drying gas temperature was 350 °C, and flow rates for the drying gas and the nebulizer gas pressure were 12 L/h and 35 psi, respectively. The capillary, fragmentor, and octupole rf voltages were set at 5000, 450, and 750, respectively. The raw data was converted to zero charge mass spectra with a maximum entropy deconvolution algorithm within the MassHunter workstation software version B.03.01. Size Exclusion Chromatography. ADC samples were analyzed offline by dual column SEC. Two 7.8 mm × 30 cm TSK gel G3000SWXL columns packed with 5 μm particles with 250 Å pores (Tosoh, JPN) were connected in series. ADC samples were separated using a mobile phase consisting of 200 mM ammonium acetate, pH 7 at a flow rate of 0.8 mL/min. UV absorbance at 280 nm was used for detection due to interfering background absorbance of the mobile phase at lower wavelengths. Hydrophobic Interaction Chromatography. vcMMAE and mcMMAF ADCs were separated on the basis of the molar ratio of antibody to drug (MR) by HIC. Butyl-NPR columns (Tosoh, JPN) with dimensions of 4.6 mm × 35 mm and 4.6 mm × 100 mm were used for separation of vcMMAE and mcMMAF ADCs, respectively. The vcMMAE mobile phase A consisted of 50 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0, and mobile phase B consisted of a mixture of 50 mM sodium phosphate, pH 7.0 and isopropanol in a 3:1 ratio. Mobile phases for separation of mcMMAF conjugates were the same except the concentration of sodium phosphate was lowered to 25 mM. Separation of vcMMAE conjugates was obtained with a linear gradient of 0−100% B over 12 min at a flow rate of 0.8 mL/min. Separation of mcMMAF conjugates necessitated a longer gradient of 15−85% B over 50 min at a flow rate of 0.4 mL/min. Quantitation of drug loaded species was obtained by injecting 40 μg and integrating the UV area of each species at 280 nm.

Figure 1. IgG1 structure associated with ADCs with a molar ratio of drug to antibody (MR) from 0 to 8. Disulfide linkages between subunits of the mAB (MR = 0) are linked to drugs in the ADC resulting in noncovalently associated 2LC-2HC drug-linked species.

populations on the basis of post-translational modifications and chemical degradations,11−13 which may be evident as coeluting or new peaks in the chromatogram thus complicating assessment of drug distribution by this technique. Finally, certain ADC characterization questions pertaining to the identity of charge and size variants present in the drug substance are best answered by measuring the mass of the intact molecule. The difficulties inherent in applying MS to molecules which are a composite of subunits bound together by covalent bonds and noncovalent interactions are nevertheless able to be overcome through the use of volatile buffers and ionization conditions which preserve native protein structure. Nondenaturing or native protein ESI-MS has been used extensively for over 2 decades to study receptor−ligand, enzyme−substrate, and protein−cofactor complexes from a variety of systems.14−16 More recently, native protein ESI-MS techniques have been used to detect very large assemblies of proteins approaching or exceeding 1000 kDa in mass17,18 and to examine the kinetics of protein unfolding.19 This report addresses the analytical gap inherent in analyzing cysteinyl-linked ADCs that has existed thus far by presenting a native, size-exclusion chromatography (SEC)-based desalting and mass measurement method. The following method can routinely be applied to the measurement of the intact mass of interchain cysteine-linked ADCs and to assess the relative distribution of drug-linked species by ion abundance.

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MATERIALS AND METHODS Antibody-Drug Conjugates. IgG1 recombinant monoclonal antibodies were expressed in CHO cells and purified according to standard procedures.20 Conjugation of mAb cysteine residues to valine-citrulline-monomethyl Auristatin E (vcMMAE) or maleimidocaproyl-monomethyl Auristatin F (mcMMAF) was carried out according to established procedures.21 Prior to mass spectrometric analysis, antibodies were deglycosylated by adding 1 μL of PNGase F (New England Biolabs, Ipswich, MA) per 100 μg of antibody or ADC and incubated at 37 °C for at least 4 h. Native-SEC Desalting. mAbs and ADCs were separated on a polyhydroxyethyl-A (PHEA) column (PolyLC, Columbia, MD) with dimensions of 2.1 mm × 200 mm and containing 5 μm particles with 300 Å pores. Buffer strength and pH optimization was carried out by varying the concentration of

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RESULTS AND DISCUSSION We hypothesized that denaturation and the ensuing dissociation of the ADC could be prevented if the analysis occurred in the absence of organic solvents and acidic ion-pairing reagents. Previous reports have described the use of ammonium acetate buffers for obtaining the intact mass of noncovalent complexes under native conditions. Other reports have specifically described the analysis of antibody monomers and multimers under native conditions in ammonium acetate buffers.23,24 For our purposes, we found that 200 mM ammonium acetate at 2844

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Figure 2. Unprocessed raw and deconvoluted MS data associated with mass measurement of deglycosylated mAb-A in denaturing and nondenaturing conditions. The unprocessed raw and deconvoluted MS data obtained in denaturing conditions are shown in panels A and C, respectively, and the unprocessed raw and deconvoluted MS data obtained in nondenaturing conditions are shown in panels B and D, respectively.

neutral pH ∼7, used in conjunction with the PHEA column provided adequate desalting of deglycosylated IgG1 mAb-A. A comparison of the raw MS data obtained from the same sample desalted in the presence of 30% acetonitrile, 0.2% formic acid to the ammonium acetate desalted sample indicated that the former conditions caused unfolding of the antibody. This observation was based on the mass range of the charge envelope, which was observed between 2000 and 4000 m/z, while the ammonium acetate desalt method yielded a charge envelope between 4800 and 6800 m/z (Figure 2, panels A and B, respectively). The ions evident between 200 and 3500 m/z in panel B are due to the presence of PNGase F, which was used for deglycosylation and the nonionic detergent Tween-80. The accuracy of the deconvoluted mass of the antibody determined by either method was within 10 ppm (Figure 2, panels C and D). The mcMMAF conjugate of mAb-A described above was also analyzed using the denaturing and nondenaturing chromatographic desalting methods. Desalting in the presence of 30% acetonitrile, 0.2% formic acid resulted in a charge envelope between 1500 and 4000 m/z (Figure 3, panel A). The deconvoluted MS was dominated by drug-linked antibody fragments, including 1LC with 1 drug, 1LC1HC with 2 drugs, 2HC with 2 drugs, 1LC2HC with 1 drug, and 2LC2HC with 0 drugs (Figure 3, panel B). There was no evidence for the presence of intact ADC-A. The same m/z region in the ammonium acetate desalted ADC-A MS

(Figure 3, panel B) was also deconvoluted and the observed dissociation of the ADC was much lower and only 1LC with 1 drug and 1LC1HC with 2 drugs were observed (Figure 3, panel D). Multiply charged ions for intact ADC-A were only observed in the raw data for the ammonium acetate desalted sample and are indicated in the bracketed area in Figure 3, panel C. The presence of dissociated ADC subunits in the nativeammonium acetate desalted MS prompted further examination of the SEC-MS method to determine when the phenomena occurred. We evaluated the desalting method as a cause of dissociation by collecting the ADC from the desalting column (Figure 4, A) and then analyzed ADC collected from the desalting column, as well as the ADC prior to desalting, by dual-column SEC. A comparison of the chromatograms from the dual-column SEC analysis of the desalted ADC and the corresponding starting material indicated that there was no increase in dissociation as a consequence of the desalting method (Figure 4). Analysis of the IgG1 mcMMAF and vcMMAE ADCs by the same ammonium acetate desalting method described above resulted in a distribution of species with masses consistent with the mAb with incorporation of 0−8 drugs. The intact mass spectra of the IgG1 mcMMAF conjugate ADC-A and the parent material, (mAb-A), are shown in Figure 5, panel A. The relative ion intensities of the individual drug loaded species were quantitated from the ion abundances associated with the 2845

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Figure 3. Unprocessed and deconvoluted MS associated with mass measurement of a deglycosylated mcMMAF conjugate ADC-A in denaturing and nondenaturing conditions. The unprocessed and deconvoluted MS data obtained in denaturing conditions is shown in panels A and C, respectively, and the unprocessed and deconvoluted MS data obtained in nondenaturing conditions is shown in panels B and D, respectively. Multiply charged ions for intact ADC-A are indicated with a bracket in panel B and deconvolution artifacts present in panels C and D are indicated with asterisks.

mAb-B, and are shown in Figure 5, panel B. Comparison of the relative amounts of ADC MR0−8 determined from the intact mass data and by HIC is shown in Figure 6 with the IgG1 mcMMAF and vcMMAE ADCs shown in panels A and B, respectively. In general, the mass measurement of the ADCs deviated from the theoretical value by a greater degree than the mAb particularly at the higher −6 and 8 drug loaded species where the difference ranged from 60 to 90 ppm, which may be due to less efficient deadducting of the higher loaded species. Analysis of noncovalent protein complexes is typically carried out using nanospray infusion which eliminates the need for heated drying gas assisted desolvation/ionization and attendant disruption of noncovalent complexes.25,26 We initially considered this approach but found that it could not be practically and routinely applied in a process development environment where an emphasis is placed on method throughput and robustness. A standard multimode ESI source was subsequently used for this work. We observed some dissociation of the ADC during mass analysis which was evident in the deconvoluted MS as LC with 1 drug and 1LC1HC with 2 drugs (Figure 3, panel D). We believe that dissociation may be occurring during ESI (postdesalt) in the liquid phase and may be due to an increase in the temperature of the ionized analyte-containing droplets by the drying gas which was heated to 350 °C to assist in desolvation.

Figure 4. SEC comparison of deglycosylated mcMMAF ADC before (starting material) and after (Post SEC desalt) native SEC desalting. Inset A is the chromatogram from the ADC desalting run. The bracketed area represents the portion of the separation that was collected for reanalysis by SEC.

deconvoluted mass spectrum. Similar results were obtained for the IgG1 vcMMAE conjugate ADC-B and the parent material, 2846

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Figure 5. Deconvoluted mass spectra for the deglycosylated mcMMAF conjugate, ADC-A, and the corresponding parent material, mAb-A, are shown in panel A. The deglycosylated vcMMAE conjugate, ADC-B, and the corresponding parent material, mAb-B, are shown in panel B. The ADC structures are shown above the corresponding deconvoluted mass in the mass spectra.

2 drugs, which is found in both denaturing and native mass analysis of ADC-A (Figure 3, panels A and B), indicates that the same charge states are observed for these species in both sets of data. We found that the most intense ions observed for 1LC1HC with 2 drugs have charge states ranging from 34+ to 39+. This indicates that 1LC1HC with 2 drugs evident in the MS is likely unfolded and may have much greater ionization efficiency than the native-intact ADC. The dominant charge state of the corresponding intact ADC with 4 drugs, which has twice the mass of the 1LC1HC-2 drug species, is 26+ and thus it is expected that the intact species would have much lower ionization efficiency relative to the denatured half-molecule. Nevertheless, the appearance of individual ADC subunits in the MS data indicates that we cannot rule out the possibility that the analytical scale intact mass analysis method is more dissociative than a classical native nano-ESI-MS procedure which would be used to analyze noncovalent protein complexes. We nevertheless believe that this is an appropriate trade-off considering the gains in efficiency and ease of use that are achievable with this approach vis a vis the classic nano-ESI-MS approach.

Denaturation of the ADC promotes dissociation into its constituent subunits which, relative to the native-intact species, compete more effectively for charge, reside closer to the surface of the ESI droplet, and are thus more apt to enter the gas phase.27,28 In spite of the dissociation, we found that when individual drug linked species were quantitated by MS and compared to results from an orthogonal method (HIC), the results were similar. It is possible that the dissociation of the ADC during ESI is occurring at levels which are sufficiently low that the relative levels of the intact drug loaded species are not significantly impacted. Along these lines, Kuprowski et al. have examined the relative intensity of native and denatured proteins in the gas phase and found that denatured proteins can have ion abundances that are 2 orders of magnitude higher than the corresponding native proteins and that this effect can result from charge competition in low ionic strength buffers as well as a greater affinity on the part of denatured proteins for the air liquid interface during ESI desolvation.29,30 They also point out that this effect is more pronounced at higher flow rates versus classic nanospray ESI. A comparison of the charge envelope of 1LC with 1 drug and 1LC1HC with 2847

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 425.527.2412. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to acknowledge Dr. Steve Alley for critical review of the manuscript and Dr. Morris Rosenberg for continuing support of this work.

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Figure 6. Relative levels of the molar ratios (MR) of mcMMAF per IgG1 ADC-A (panel A) and vcMMAE per IgG1 ADC-B (panel B) as determined by MS based quantitation from the deconvoluted mass spectra and by UV integration of the species separated by HIC.

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REFERENCES

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CONCLUSIONS

From a practical standpoint, the analytical scale native intact mass analysis method offers several key advantages over other methods that are used to assess interchain cysteinyl-linked ADCs. The mass data obtained using the method are well resolved and sufficient for confirmation of the identity of the ADCs. Additionally, the relative distribution of the drug loaded species assessed by their ion abundance in the deconvoluted mass spectrum does not vary significantly from the distribution of drug loaded species determined by off-line HIC methods. Moreover, the maximum cycle necessary for the analysis of a given sample using the described method is 10 min, which compares favorably to typical HIC methods where separation and quantitation of individual species can take up to 1 h or more. While we have leveraged this approach to document the first reported intact mass measurement of an ADC with drugs linked to interchain cysteine residues, the aforementioned attributes of the method indicate that it can be readily adopted in a process development environment and run on a routine basis. 2848

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