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High-resolution accurate-mass mass spectrometry enables in-depth characterization of in vivo biotransformations for intact antibody-drug conjugates Jintang He, Dian Su, Carl K. Ng, Luna Liu, Shang-Fan Yu, Thomas H Pillow, Geoffrey Del Rosario, Martine Darwish, Byoung-Chul Lee, Rachana Ohri, Hongxiang Zhou, Xueji Wang, Jiawei Lu, Surinder Kaur, and Keyang Xu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Apr 2017 Downloaded from http://pubs.acs.org on April 21, 2017

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Analytical Chemistry

High-resolution accurate-mass mass spectrometry enables in-depth characterization of in vivo biotransformations for intact antibody-drug conjugates Jintang He,*,1 Dian Su,1 Carl Ng,1 Luna Liu,1 Shang-Fan Yu,1 Thomas H. Pillow,1 Geoffrey Del Rosario,1 Martine Darwish,1 Byoung-Chul Lee,1 Rachana Ohri,1 Hongxiang Zhou,2 Xueji Wang,2 Jiawei Lu,2 Surinder Kaur,1 and Keyang Xu*,1 1

Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, United States

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Wuxi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China

Corresponding Authors: *Email [email protected]; phone +1 650-225-6956; fax +1 650-225-1998. *Email [email protected]; phone +1 650-225-4525; fax +1 650-225-1998.

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ABSTRACT

Antibody-drug conjugates (ADCs) represent a promising class of therapeutics for the targeted delivery of highly potent cytotoxic drugs to tumor cells to improve bioactivity while minimizing side effects. ADCs are composed of both small and large molecules, and therefore have complex molecular structures. In vivo biotransformations may further increase the complexity of ADCs, representing a unique challenge for bioanalytical assays. Quadrupole time-of-flight mass spectrometry (Q-TOF MS) with electrospray ionization has been widely used for characterization of intact ADCs. However, interpretation of ADC biotransformations with small mass changes, for the intact molecule, remains a limitation due to the insufficient mass resolution and accuracy of Q-TOF MS. Here, we have investigated in vivo biotransformations of multiple site-specific THIOMABTM antibody-drug conjugates (TDCs), in the intact form, using a high-resolution, accurate-mass (HR/AM) MS approach. Compared with conventional Q-TOF MS, HR/AM Orbitrap MS enabled more comprehensive identification of ADC biotransformations. It was particularly beneficial for characterizing ADC modifications with small mass changes such as partial drug loss and hydrolysis. This strategy has significantly enhanced our capability to elucidate ADC biotransformations and help understand ADC efficacy and safety in vivo.


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INTRODUCTION

Antibody-drug conjugates (ADCs) are currently being studied in clinical trials for a wide range of hematological and solid tumor indications, with over 40 molecules under investigation and two approved by the US FDA to date.1,2 ADCs are designed for targeted delivery of cytotoxic drugs to tumor cells to improve therapeutic efficacy with minimal systemic toxicity.3-5 The drug is conjugated to the antibody via a linker, and the conjugation reaction typically results in a heterogeneous mixture with a range of drug-to-antibody ratios (DARs).6 For example, conjugation through lysine residues often leads to a distribution of DARs ranging from 0 to 9, while conjugation through reduced interchain disulfide bonds results in mainly even numbered DARs 0, 2, 4, 6 and 8. To generate homogeneous ADCs, Junutula and co-workers have recently developed a new method that allows site-specific conjugation through engineered cysteines.7-9

The conjugates generated by this method are also referred to as

THIOMABTM antibody-drug conjugates (TDCs). ADCs have complex structures as they are a composite of both small and large molecules. In addition, ADCs often undergo biotransformations over time in vivo,6,10-13 such as linker deconjugation, drug loss, partial drug loss, adduct formation, hydrolysis and other modifications. These structural changes further increase the complexity of ADCs. It is important to characterize the identities of various ADC biotransformations because this not only provides insights into metabolism and catabolism of ADCs in vivo, but also improves our understanding of bioactivity changes of ADCs and guides the design of conjugates. Characterization of ADC biotransformations represents a unique challenge for bioanalytical assays.6,14-16 Ligand-binding assays are routinely used to measure total antibody concentration but are unable to distinguish between different ADC species where the linker or drug has undergone distinct structural changes.6,17 LC-MS/MS has been used to quantify the antibody or the drug of an ADC by selective monitoring the signature peptides or the small molecule moiety.18-22 However, the assay only focuses on a small part of the ADC with predefined structure. To better understand ADC 3 ACS Paragon Plus Environment


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biotransformations, it is ideal to analyze the ADC in the intact form with minimal treatment, in order to preserve its structural information as much as possible. Our group has previously developed an affinity capture LC-MS technique and successfully characterized linker-drug deconjugation for a variety of intact TDCs.10,12,14 Quadrupole time-of-flight (Q-TOF) MS is routinely used in this approach to efficiently resolve multiply charged ions of high molecular weight intact ADCs.10,14,23-25 However, there is evidence that the resolution offered by a TOF instrument may not be sufficient to interrogate ADCs with complex structures. For example, when analyzing an intact maleimide linked MMAF conjugate using Q-TOF MS, a series of broad peaks were observed after 10 days post-dose.12 Each broad peak is very likely to represent a mixture of multiple DAR species that were not resolved by Q-TOF MS. Our group recently reported a custom-designed affinity capture LC-MS F(ab’)2 assay for TDCs with conjugation sites located in the Fab region, where the Fc domain was removed by IdeS digestion. The smaller size analytes, with the size reduced to ~ 100 kDa, result in improved mass resolution and more accurate peak assignment.26 However, we also realize that there is a certain limitation associated with this approach, which is that it is not applicable for TDCs whose conjugation sites are located in the Fc region or ADCs with random lysine conjugation. Recent reports indicate that Orbitrap instruments offer greater resolution for analysis of a mixture of monoclonal antibodies27 and standard ADC reagents28,29. The latest advancement in Orbitrap technology could also provide additional benefits for exploring ADC biotransformations in vivo. In this work, we have demonstrated the use of a high-resolution, accurate-mass (HR/AM) Orbitrap MS approach for characterizing in vivo biotransformations of intact ADCs. Compared with conventional Q-TOF MS, HR/AM Orbitrap MS provided enhanced resolving power to allow more comprehensive identification of species newly formed in circulation, which is critical to better understand the ADC metabolism/catabolism and functional changes in vivo. This new workflow can be critical for adequately identifying biotransformations with small mass changes (e.g., deacetylation) at the level of intact ADCs, which could be challenging with the existing affinity capture LC-MS setup.14 This new approach is with


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a broader applicability, suitable for ADCs with site specific conjugations in both Fab and Fc regions, as well as for ADCs with random lysine conjugations (data to be reported separately later).

EXPERIMENTAL SECTION Animal Plasma Samples. All animal studies were carried out in compliance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Genentech, Inc. CB17 SCID mice or Sprague-Dawley rats (Charles Rivers Laboratories, Reno, NV) were administered a single intravenous injection of ADC through the tail vein. All conjugates used in this work were TDCs. Blood samples were collected into tubes containing lithium heparin via retro-orbital bleeds and used to derive plasma. The plasma samples were stored at -70 °C until the following analysis. Affinity Capture. ADCs were selectively extracted from plasma samples by affinity capture as described previously.12,14 Briefly, human Her2 or CD22 extracellular domain (ECD) was biotinylated and immobilized onto Dynabeads M-280 Streptavidin (Invitrogen, Carlsbad, CA) in a 96-well plate, and then the ECD-bead system was used to capture ADCs by incubating with plasma samples for 2 hours at room temperature. The captured ADCs were then washed with HBS-EP buffer (10 mM Hepes [pH 7.4], 150 mM NaCl, 3.4 mM ethylenediaminetetraacetic acid [EDTA], 0.005% Surfactant P20) (GE Healthcare, Piscataway, NJ) and deglycosylated using N-Glycanase (Prozyme, San Leandro, CA) at 37 °C overnight. After extensive washing of the beads with HBS-EP, water and 10% acetonitrile, the ADC analytes were eluted using 30% acetonitrile with 1% formic acid. A KingFisher 96 magnetic particle processor (Thermo Fisher Scientific, Waltham, MA) was used to mix, wash, gather, and transfer the paramagnetic beads in the above steps. Reversed Phase Liquid Chromatography. Chromatographic desalting and separation of ADCs were performed on a nanoACQUITY UPLC® system (Waters Corporation, Milford, MA) equipped with a PS-DVB monolithic column (500 µm i.d. x 5 cm) (Thermo Fisher Scientific, Waltham, MA). Five microliters of the ADC analyte were loaded onto the column and separated from interference ACS Paragon Plus Environment

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species with a gradient of mobile phase A (0.1% formic acid) and mobile phase B (acetonitrile, 0.1% formic acid) at a flow rate of 15 µL/min. The gradient was 0%-40% B in 4 min, 40% B for 3 min, and 40-100% B in 1.5 min. Intact ADC Analysis by Q-TOF MS. Following chromatographic separation, the ADC samples were analyzed on a TripleTOF 5600 mass spectrometer (AB Sciex, Concord, ON) equipped with a DuoSprayTM ion source. The instrument was operated in positive ion mode. The main parameters were set as follows: ionspray voltage floating, 5 kV; declustering potential, 250 V; collision energy, 20 V. TOF-MS spectra were extracted from the total ion chromatograms (TICs) and deconvoluted using the Bayesian Protein Reconstruct algorithm incorporated in BioPharmaViewTM 2.0 software (AB Sciex, Concord, ON), and the average DAR was calculated based on the peak areas of different DAR species 14,26

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Intact ADC Analysis by Orbitrap MS. The ADC samples were also analyzed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an Ion Max API source. For intact ADCs, the spectra were acquired using a Full MS method at a resolution setting of 17,500 and the key parameters were optimized as follows: spray voltage, 3.2 kV; sheath gas flow rate, 8; S-lens RF level: 100; in-source CID, 100 ev; AGC target, 3 x 106; maximum injection time, 150 ms; microscans, 10. Full MS spectra were extracted from the TICs and deconvoluted using the ReSpectTM algorithm incorporated in Protein Deconvolution 4.0 software (Thermo Fisher Scientific, Bremen, Germany). The mass tolerance was set as 25 ppm. The average DAR was calculated based on the peak intensity of different DAR species. Analysis of Reduced ADCs by Orbitrap MS. For analysis of light and heavy chain species, ADCs were reduced by incubation at 37 °C for 40 min with 10 mM tris(2-carboxyethyl)phosphine (TCEP). Chromatographic separation of light and heavy chains was performed on a nanoACQUITY UPLC® system equipped with a PS-DVB monolithic column (200 µm i.d. x 25 cm) (Thermo Fisher Scientific, Waltham, MA) using a gradient of mobile phase A (0.1% formic acid) and mobile phase B (acetonitrile, 0.1% formic acid) at a flow rate of 3 µL/min. The gradient was 0%-25% B in 4 min, 25% ACS Paragon Plus Environment

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B for 3 min, 25-35% B in 22 min, and 35-60% B in 3 min. Reduced ADCs were analyzed on a Q Exactive Plus mass spectrometer, and deconvolution was performed using Protein Deconvolution 4.0.

RESULTS AND DISCUSSION HR/AM MS and conventional TOF MS displayed similar DAR distribution for ADCs with minimal biotransformation. To compare the performance of Orbitrap and TOF MS for characterizing intact ADCs, we first analyzed a disulfide-linked pyrrolobenzodiazepine (PBD) dimer TDC with relatively simple DAR distribution and minimal biotransformation. The TDC was administered to mice intravenously at 5 mg/kg and the plasma samples were harvested at 3 days post-dose. The TDC was then captured from plasma and analyzed by LC-MS on both TripleTOF 5600 (Figure 1a) and Q Exactive plus (Figure 1b). DAR1 and DAR0 species were observed by day 3. Based on their molecular masses, they were formed from DAR2 via disulfide exchange. The comparison between Q-TOF MS and HR/AM Orbitrap MS showed that the DAR distribution was highly consistent on the two MS systems (Figure 1). In addition, the calculation of average DAR resulted in the same value 1.5 for TOF and Orbitrap data. We have analyzed multiple ADCs with relatively simple mass peak profiles and obtained similar DAR distribution profiles (data not shown). This indicates that HR/AM Orbitrap MS and TOF MS have comparable performance for analyzing ADCs with limited biotransformations such as deconjugation. HR/AM MS enabled more comprehensive identification of ADC biotransformations in vivo. To investigate complex biotransformations of ADCs, we also analyzed the same disulfide-linked PBD conjugate at 10 days post-dose because more structural changes were expected to take place in vivo over time. The overall DAR distributions on Q-TOF (Figure 2a) and HR/AM Orbitrap (Figure 2b) appeared to be similar at the first glance. However, a more detailed view of the HR/AM spectrum indicated additional biotransformation information. As shown in the zoomed-in mass spectrum, two DAR1 species (P5 and P6) with a mass difference of ~ 20 Da were well resolved by Orbitrap (Figure 2c). ACS Paragon Plus Environment

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Based on the molecular masses, we identified P5 as a cysteine adduct formed by disulfide exchange, i.e., deconjugation of the linker-drug followed by the addition of a cysteine to the antibody, of a glycated DAR2 species (P2), and P6 as a glutathione adduct formed by disulfide exchange of the original DAR2 conjugate (P1). In contrast, these two DAR1 species were not resolved on Q-TOF due to the lower resolution. Instead they merged into one peak TP1 (Figure 2a). Similarly, two DAR0 species (P9 and P10) with a mass difference of ~ 20 Da were resolved by Orbitrap (Figure 2c) but not resolved by QTOF (TP2, Figure 2a). We have for the first time resolved P5/P6 or P9/P10 species. To the best of our knowledge, the identities of these biotransformations have not been previously reported. The observations here indicate that HR/AM MS resolves ADC molecular masses for biotransformations having small mass differences and thereby allows more accurate structural elucidation. A total of four DAR1 (P4-P7) and four DAR0 (P8-P11) species were identified by HR/AM MS, where P4 and P8 were the most abundant DAR1 and DAR0 species, respectively (Figure 2c). Both P4 and P8 were cysteine adducts of the original DAR2 conjugate (P1), and were significantly more abundant than their corresponding glutathione adducts P6 and P10. The abundance of cysteine and glutathione adducts appears to correlate well with the concentration of the corresponding reactive thiol in plasma, where reduced cysteine has a significantly higher concentration than reduced glutathione.30-32 HR/AM MS enabled in-depth characterization of functional group modifications for intact ADCs, thereby providing insightful information to explain in vivo efficacy outcomes. ADCs can potentially undergo partial drug loss in circulation, which could lead to loss of function. As an example, here we explored biotransformations resulting from partial drug loss of two anti-CD22 MC-vc-PAB tubulysin M TDC variants (i.e., tubulysin TDC1 and TDC2) and the impact of the structural changes on in vivo efficacy. Tubulysin M was conjugated through thiol-maleimide chemistry (Figure 3a) and administered to mice intravenously at 1 mg/kg. Plasma samples were collected at 1 and 4 days post-dose and the conjugate in each sample was analyzed by affinity capture LC-MS on both HR/AM Orbitrap and Q-TOF (Figure S1). A detailed view of the deconvoluted spectra (mass range 148,600 to 149,600 Da)


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suggests tubulysin TDC1 underwent rapid deacetylation in vivo (Figure 3b-g). The HR/AM data showed that after one day two new species were formed via loss of one or two acetyl groups from the original DAR2 conjugate, respectively, and they were baseline resolved by Orbitrap (Figure 3c). It has been reported that deacetylation results in loss of potency for tubulysin33. Therefore, we considered the two new species as DAR1 and DAR0, respectively. The tubulysin acetate appeared to be very unstable in mouse in vivo, with ~ 100% loss of the acetyl groups within four days (Figure 3d). Similar structural changes were also observed for the glycated conjugates (Figure 3c). In contrast, Q-TOF MS did not have the capability to resolve the deacetylated DAR1 and DAR0 species in the intact form (Figure 3f). Indeed, the overall DAR distribution patterns of the three time points 0 hr, 1 day and 4 days looked very similar on Q-TOF (Figure S1). Without carefully checking the molecular mass for each peak across all time points, the TOF MS data could potentially have led to a misleading conclusion that this TDC was stable in vivo. In addition, the TOF MS data do not allow us to calculate the average DAR values and accurately assess the in vivo stability of this TDC because the deacetylated DAR1 and DAR0 species were not resolved. The example described here clearly demonstrated the advantages of HR/AM MS for analyzing partial drug loss of intact ADCs. To gain a better understanding of the impacts of deacetylation on in vivo efficacy, we also analyzed a more stable tubulysin TDC2 by affinity capture LC-MS (Figure S2). Compared to tubulysin TDC1, tubulysin TDC2 had much lower deacetylation rate as displayed in the detailed view of the mass spectra (Figure S3). After one day, the dominant DAR species was still the acetylated DAR2 (Figure S3b), and even after four days, about 50% of all DAR species still carried one acetyl group (Figure S3c). Similar to the observation on tubulysin TDC1, the deacetylated DAR1 and DAR0 species for tubulysin TDC2 were well resolved by HR/AM Orbitrap MS but not by Q-TOF MS (Figure S3). Due to the enhanced resolution, the HR/AM data allowed us to calculate the average DAR values and assess the in vivo stability of the two tubulysin conjugates. Tubulysin TDC2 was confirmed to be more stable than tubulysin TDC1 over time (Figure 4a). Minimal linker-drug deconjugation was observed for both conjugates (Figures S1 and S2), and therefore loss of active drug was primarily due to deacetylation. ACS Paragon Plus Environment

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Note that the in vivo stability of these conjugates correlated well with the in vivo efficacy. The more stable tubulysin TDC2 showed greater efficacy with tumor regression in a human non-Hodgkin lymphoma xenograft in mice, compared to the instable tubulysin TDC1 resulting in only modest tumor growth delay (Figure 4b). The observations here indicate that HR/AM MS not only improves our capability to identify structural changes of payloads for intact ADCs, but also allows us to predict or explain the in vivo efficacy outcomes more accurately. HR/AM MS provided new insights into modifications of intact ADCs with small mass changes such as hydrolysis/hydration. It is well known that maleimide linkers are susceptible to hydrolysis at certain conjugation sites which results in a relatively small mass change (~18 Da) for intact ADCs.12,34,35 Similarly, depending on their chemical structures, drugs are expected to also undergo hydrolysis or hydration in circulation. Our prior experience suggests that conventional Q-TOF MS does not have the capability to resolve the new hydrolyzed version from native ADCs. Here we investigated the hydrolysis/hydration of ADCs using an anti-Her2 LC-V205C PBD dimer TDC as a model, which was administered to Sprague-Dawley rats and analyzed at four days post-dose. According to the molecular structure, there are several potential hydrolysis/hydration sites for the linker-drug. However, TOF MS spectrum showed several broad peaks with a high baseline, which made it challenging to detect any hydrolysis/hydration (Figure 5a). In contrast, HR/AM Orbitrap was able to resolve more DAR species. As shown in the inset of Figure 5b, an additional peak next to the original DAR2 peak was detected and it has a mass increase of ~30 Da, which could represent a mixture of several hydrolyzed ADC species. To confirm this hypothesis, we further reduced the TDC captured from rat plasma and analyzed it on Q Exactive plus. Hydrolysis/hydration was clearly detected for light chain attached with the linker-drug but not for naked light chain (Figure 5c) or naked heavy chain (Figure S4) as expected. This indicates that the hydrolysis/hydration observed here was attributed to the linker-drug rather than the antibody. Indeed, three newly formed light chain species due to hydrolysis/hydration were simultaneously detected (Figure 5c). When examining the structure of the linker-drug, we found one potential hydrolysis site


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(i.e., the succinimide ring) and two potential hydration sites (i.e., the two PBD imines). The three sites were highlighted in Figure 5d. As far as we know, this is the first report that up to three sites could undergo hydrolysis/hydration for this class of molecules. Based on the LC-MS data for the reduced PBD conjugate, we could theoretically detect six peaks adjacent to the intact DAR2 TDC resulting from hydrolysis/hydration. However, only one additional peak was detected (Figure 5b). Indeed, this LC-V205C PBD dimer TDC may be a heterogeneous mixture of conjugates with different levels of hydrolysis/hydration across multiple sites. Although the resolution of Orbitrap MS was not sufficient to unambiguously identify the modifications by analyzing the intact ADC, the observation of the additional peak guided us to reduce the conjugate to confirm the identity. Therefore, the additional peak resolved by Orbitrap MS represents a significant improvement. Although the resolution of the HR/AM data was substantially better than that of the Q-TOF data where no hydrolysis/hydration was detected, it seems that there is still a lot of room for further improvement. New advancements in high-resolution mass spectrometry technologies, as well as in sample preparation techniques, would further enhance our capability to elucidate ADC biotransformations.

CONCLUSIONS In this work we have demonstrated that HR/AM MS enabled us to gain new insights into ADC biotransformations in vivo. When analyzing ADCs with relatively simple DAR distribution profiles and minimal biotransformation, HR/AM Orbitrap MS agreed well with conventional Q-TOF MS. However, when analyzing ADCs that undergo complex structural changes in vivo, HR/AM MS provided additional biotransformation information that was not available by TOF MS. Analysis of a disulfidelinked PBD TDC at 10 days post-dose in mouse by HR/AM Orbitrap MS resulted in the identification of eight distinct biotransformations (P4-P11 in Figure 2c) in vivo, while four of them were not accurately identified by Q-TOF MS due to its lower resolution and mass accuracy. HR/AM MS also enabled indepth characterization of partial drug loss for intact ADCs with a small mass change. For example, it ACS Paragon Plus Environment

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was able to resolve different DAR species for tubulysin M conjugates resulting from deacetylation, thereby allowing us to evaluate the in vivo stability of the conjugates and get a better understanding of in vivo efficacy. Using HR/AM MS, we have also investigated hydrolysis/hydration, which is a common modification for many ADCs in vivo. We detected a new species for an intact PBD conjugate potentially formed by hydrolysis/hydration. By analyzing the reduced conjugate, we found three potential hydrolysis/hydration sites in the linker-drug. Due to the enhanced resolution and mass accuracy, HR/AM Orbitrap MS greatly increases our capability to identify ADC biotransformations, especially those with small mass changes. Improving our ability to understand ADC metabolism or catabolism in vivo helps provide greater insights into the safety and efficacy of ADCs in vivo. Since the mass resolution was improved at the level of intact ADCs, the strategy is potentially suitable for analyzing most ADCs regardless of the location of conjugation sites.


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Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional information about deconvoluted spectra and DAR distribution (PDF).


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(13) Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. A.; Cerveny, C. G.; Chace, D. F.; DeBlanc, R. L.; Gearing, R. P.; Bovee, T. D.; Siegall, C. B.; Francisco, J. A.; Wahl, A. F.; Meyer, D. L.; Senter, P. D. Nat Biotechnol 2003, 21, 778-784. (14) Xu, K.; Liu, L.; Saad, O. M.; Baudys, J.; Williams, L.; Leipold, D.; Shen, B.; Raab, H.; Junutula, J. R.; Kim, A.; Kaur, S. Anal Biochem 2011, 412, 56-66. (15) Saad, O. M.; Shen, B. Q.; Xu, K.; Khojasteh, S. C.; Girish, S.; Kaur, S. Bioanalysis 2015, 7, 15831604. (16) Alley, S. C.; Anderson, K. E. Curr Opin Chem Biol 2013, 17, 406-411. (17) Rago, B.; Clark, T.; King, L.; Zhang, J.; Tumey, L. N.; Li, F.; Barletta, F.; Wei, C.; Leal, M.; Hansel, S.; Han, X. Bioanalysis 2016, 8, 2205-2217. (18) Xu, K.; Liu, L.; Maia, M.; Li, J.; Lowe, J.; Song, A.; Kaur, S. Bioanalysis 2014, 6, 1781-1794. (19) Shen, B. Q.; Bumbaca, D.; Saad, O.; Yue, Q.; Pastuskovas, C. V.; Khojasteh, S. C.; Tibbitts, J.; Kaur, S.; Wang, B.; Chu, Y. W.; LoRusso, P. M.; Girish, S. Curr Drug Metab 2012, 13, 901-910. (20) Wang, J.; Gu, H.; Liu, A.; Kozhich, A.; Rangan, V.; Myler, H.; Luo, L.; Wong, R.; Sun, H.; Wang, B.; Vezina, H. E.; Deshpande, S.; Zhang, Y.; Yang, Z.; Olah, T. V.; Aubry, A. F.; Arnold, M. E.; Pillutla, R.; DeSilva, B. Bioanalysis 2016, 8, 1383-1401. (21) Sanderson, R. J.; Nicholas, N. D.; Baker Lee, C.; Hengel, S. M.; Lyon, R. P.; Benjamin, D. R.; Alley, S. C. Bioanalysis 2016, 8, 55-63. (22) Liu, A.; Kozhich, A.; Passmore, D.; Gu, H.; Wong, R.; Zambito, F.; Rangan, V. S.; Myler, H.; Aubry, A. F.; Arnold, M. E.; Wang, J. J Chromatogr B Analyt Technol Biomed Life Sci 2015, 1002, 5462. (23) Grafmuller, L.; Wei, C.; Ramanathan, R.; Barletta, F.; Steenwyk, R.; Tweed, J. Bioanalysis 2016, 8, 1663-1678. (24) Hengel, S. M.; Sanderson, R.; Valliere-Douglass, J.; Nicholas, N.; Leiske, C.; Alley, S. C. Anal Chem 2014, 86, 3420-3425. (25) Birdsall, R. E.; Shion, H.; Kotch, F. W.; Xu, A.; Porter, T. J.; Chen, W. MAbs 2015, 7, 1036-1044. ACS Paragon Plus Environment

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Acknowledgments: We would like to thank Kathy Kozak, Jack Sadowsky, Mehraban Khosraviani, Violet Lee, Rebecca Rowntree, Douglas Leipold and Peter Dragovich for scientific discussions. Conflict of Interest Disclosure: The authors declare no competing financial interest.


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Figure 1. Drug-to-antibody ratio distribution for a disulfide-linked PBD-dimer TDC. Mice were dosed intravenously with 5 mg/kg of the conjugate. Plasma was harvested at 3 days post-dose and the conjugate was analyzed by affinity capture LC-MS on both TOF (a) and Orbitrap (b) instruments.


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Figure 2. HR/AM MS enables more comprehensive identification of in vivo biotransformations for an anti-Her2 PBD-dimer TDC. Mouse plasma was harvested at 10 days post-dose and the TDC was analyzed by affinity capture LC-MS on both TOF (a) and Orbitrap (b) instruments.

Additional

biotransformations were observed from a detailed view of the HR/AM spectrum (c). P1: DAR2; P2: DAR2 + Hex; P3: DAR2 + 2Hex; P4: DAR2 - LD + Cys; P5: (DAR2 + Hex) - LD + Cys; P6: DAR2 LD + GSH; P7: (DAR2+ Hex) - LD + GSH; P8: DAR2 - 2LD + 2Cys; P9: (DAR2+ Hex) - 2LD + 2Cys; P10: DAR2 - 2LD + Cys + GSH; P11: (DAR2+ Hex) - 2LD + Cys + GSH. Hex represents hexose, LD represents linker-drug, Cys represents cysteine, and GSH represents glutathione. DAR2, DAR1 and DAR0 species were labeled in red, green and purple, respectively. TP1 is a DAR1 peak and TP2 is a DAR0 peak on the Q-TOF spectrum. ACS Paragon Plus Environment

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Figure 3. Characterization of deacetylation for anti-CD22 MC-vc-PAB Tubulysin M TDC1. SCID mice were dosed intravenously with 1 mg/kg of anti-CD22 MC-vc-PAB Tubulysin M TDC1 (a). Plasma samples were harvested at 1 and 4 days post-dose and the conjugate was analyzed by HR/AM Orbitrap MS (b, c and d) and TOF MS (e, f and g). DAR2, DAR1 and DAR0 species were labeled in red, green and purple, respectively.


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Figure 4. Impact of deacetylation rate on in vivo efficacy of anti-CD22 MC-vc-PAB tubulysin M. (a) In vivo stability of tubulysin TDC1 and TDC2. The tubulysin conjugates were administered to SCID mice intravenously at 1 mg/kg. In vivo stability was determined using affinity capture HR/AM Orbitrap MS. The average DAR was calculated based on peak intensity of different DAR species and normalized to the starting DAR. (b) In vivo efficacy of the tubulysin conjugates in BJAB-luc human lymphoma xenograft model in mice. SCID mice bearing lymphoma xenografts (n=8/group) were dosed with vehicle, tubulysin TDC1 or TDC2. Changes in tumor volumes over time for each treatment group are depicted as cubic spline fits generated via linear mixed effects analysis of log-transformed volumes as previously described 36.


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Figure 5. Hydrolysis/Hydration of an anti-Her2 LC-V205C PBD dimer TDC. Sprague-Dawley rats were dosed intravenously with 5 mg/kg of this TDC. Plasma was harvested 4 days post-dose and the conjugate in each plasma sample was analyzed by affinity capture LC-MS on both TOF (a) and Orbitrap (b) instruments. Following analysis of the intact TDC, the leftover sample was reduced with TCEP and analyzed by LC-MS (c). Three potential hydrolysis/hydration sites were highlighted in green (d). L represents light chain, and LD represents linker- drug.


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