MS Assay for Plasma Stability and

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Technical Note Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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A Two-Step Immunocapture LC/MS/MS Assay for Plasma Stability and Payload Migration Assessment of Cysteine−Maleimide-Based Antibody Drug Conjugates Linlin Dong,* Chao Li,† Charles Locuson, Susan Chen, and Mark G. Qian Department of Drug Metabolism and Pharmacokinetics, Takeda Pharmaceuticals International, Inc., 35 Landsdowne Street, Cambridge, Massachusetts 02139, United States S Supporting Information *

ABSTRACT: Plasma stability assessment under physiological temperature is an essential step for developing and optimizing antibody drug conjugate (ADC) molecules, especially those with cleavable linkers. The assessment of plasma stability often requires monitoring multiple analytes using a combination of bioanalytical assays for free payloads, conjugated payloads (or conjugated antibodies), total antibodies, and payloads that have migrated from antibodies to plasma constituent proteins. Bioanalytical assays are needed in early drug discovery to quickly screen diverse ADC candidates of different antibody constructs, linker variants, and antibody anchor sites. To improve the sensitivity and selectivity of LC/MS/MS-based assays for the assessment, immunocapture has been widely used for extracting ADCs and unconjugated antibodies from plasma samples. In this study, a novel two-step immunocapture LC/MS/MS assay was described to allow the quantification of conjugated payloads, total antibodies, and migrated payloads forming adducts with albumin in the plasma samples for stability assessment. A target antigen immobilized on magnetic beads was used to exhaustively extract the ADC and antibody-associated species. The remaining supernatant was then extracted further with anti-albumin beads for recovering the albumin-associated adducts for quantification. The method was optimized for higher efficiency and cost-effectiveness using microwave enhanced papain-based enzymatic cleavage for measuring conjugated payloads of ADCs and lysyl endopeptidase cleavage in the total antibody assay. A maleimide linker-based ADC with a proprietary payload, TAK-001, was used to demonstrate the streamlined workflow of the ADC stability assessment. The method could provide valuable evaluation of the stability of the ADC as well as the quantitative assessment of the albumin adducts formed from the linker-payload migration in mouse and human plasma. Furthermore, the method should be readily adaptable for other ADCs using thiol−maleimide conjugation chemistry.

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(e.g., cathepsin B) after the internalization to effect a direct kill of tumor cells. Given that brentuximab vedotin and many similar ADCs are designed to release payloads via hydrolysis, there is a risk that some cleavable linkers could prematurely release payloads in circulation, prior to entering the target tissue and cells. Hence, for ADCs using the cleavable payload technology, in vitro plasma stability in early ADC drug discovery is normally conducted to assess this risk. Generation of free payloads from a cleavable linker in plasma is easily monitored by LC/MS. With a few more steps, the remaining ADCs in plasma can be immunocaptured, and the residual payload may be cleaved using chemical or enzymatic approaches to calculate the amount of antibody-conjugated payloads. However, thiol−maleimide-based ADCs in particular may also undergo linker-payload deconjugation through a thiolexchange process primarily with reactive cysteines on the plasma albumin,1,7−11 which could result in not only the loss of payload from ADCs but theoretically could also give rise to immunotoxicity from the new payload albumin adducts formed.

ntibody drug conjugates (ADCs) are hybrid molecules that consist of monoclonal antibodies conjugated with small molecule payloads through a linker. Most ADCs are designed to deliver highly potent cytotoxic payloads into targeted tumor cells in the body. The intention of making such hybrid molecules is to trigger tumor cell death while limiting off-target toxicity. This is accomplished via antibody targeting of specific cell surface antigens followed by ADC internalization and cytotoxic payload release into the target cells. Currently, four ADCs have been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA), including brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, and gemtuzumab ozogamicin.1−5 Among the four ADCs, brentuximab vedotin and trastuzumab emtansine used a cysteine or lysine anchor with maleimide conjugation technology to link antibodies and payloads through either a cleavable or noncleavable bridge.6 The success of maleimide-based conjugation chemistry has generated wide enthusiasm in applying the technology to many prospective monoclonal antibody and toxin combinations in both preclinical and clinical stages.1,7 In brentuximab vedotin, the cleavable payload, monomethyl auristatin E (MMAE), can be liberated by lysosomal enzymes © XXXX American Chemical Society

Received: February 9, 2018 Accepted: April 13, 2018

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DOI: 10.1021/acs.analchem.8b00694 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry

Free Payload Plasma Stability. In vitro stability samples of TPP (1 μM) were prepared in both mouse and human plasma through diluting 10 mM of the TPP stock solution in DMSO 10 000-fold. An aliquot of 500 μL from each preparation was taken immediately and stored at −80 °C as the time zero (T0) sample. Two additional 500 μL aliquots from each preparation were incubated for up to 6 days at 37 °C. Aliquots were removed from the 37 °C incubator and immediately transferred to a −80 °C freezer on day 3 and day 6 for stability assessment afterward. A detailed sample preparation procedure and LC/MS/MS conditions for free or deconjugated payload (TPP cleaved off from the ADC) analysis are described in the Supporting Information. The lower limit of quantitation (LLOQ) for TPP assay was 0.05 ng/mL. ADC Plasma Stability. In vitro stability samples of TAK001 (10 μg/mL) were prepared in both mouse and human plasma through diluting 1 mg/mL of TAK-001 stock solution 100-fold. An aliquot of 200 μL from each preparation was taken immediately and stored at −80 °C as the T0 sample. Four additional 200 μL aliquots were taken from each preparation and incubated for up to 6 days at 37 °C. Aliquots were removed from the 37 °C incubator and immediately transferred to a −80 °C freezer at 24, 48, 72, and 144 h for subsequent stability assessment. A detailed single-step immunocapture sample preparation procedure and LC/MS/MS conditions for the total antibody and conjugated payload analysis are described in the Supporting Information. The LLOQ of the total antibody assay was 0.2 μg/mL and the LLOQ of the conjugated TPP assay was 0.2 ng/mL. Payload Deconjugation Using Papain vs Cathepsin B. In Group 1, cathepsin B (10 μL of 40 μg/mL) solution was spiked into a mixture of 10 μL of TAK-001 (100 μg/mL) and 100 μL of NaOAc buffer (100 mM, pH = 5). Alternatively, in Groups 2 and 3, 10 μL of activated papain (20 and 80 mg/mL, respectively) was spiked into a mixture of 10 μL of TAK-001 (100 μg/mL) and 100 μL of papain activation solution (pH = 6.5, containing 100 mM of NaOAc, 10 mM of EDTA disodium, and 2 mM of L-cysteine). Duplicate samples were incubated at 37 °C for 40 min on the REDS (a power output of 400 W was used throughout the work). At the end of the incubation, the sample was immediately acidified by adding 10 μL of 20% TFA to stop the reaction, and then, protein was precipitated using acetonitrile as detailed in conjugated payload analysis of the Supporting Information. Optimization of Digestion Time for TAK-001 in Mouse Plasma. Per 100 μL of papain activation buffer, 10 μL of TAK001 freshly spiked mouse plasma sample (100 μg/mL) was added. Samples were then mixed well, and 20 μL of activated papain solution (10 mg/mL) was added to them. For all time points other than T0, TAK-001 samples were incubated at 37 °C under the microwave condition for up to 75 min (n = 12, duplicates at each time point). Two independent aliquots were removed from the REDS at 5, 15, 30, and 45 min. Meanwhile, at 45 min, an additional aliquot of 20 μL of freshly activated papain solution was added to the 60 min and 75 min samples, which were collected later as scheduled. All samples were immediately acidified upon removal from REDS by adding 10 μL of 20% TFA to stop the reaction and followed by the same protein precipitation procedure used for papain and cathepsin B deconjugation comparison. Quantitation of Plasma Albumin Linker-Payload Adducts/Other Potential Protein Payload Adducts (Two-Step Immunocapture). An aliquot of 10 μL of each

Analytically, the thiol-exchange process complicates the interpretation of stability screening. Monitoring free payload in plasma can be used to assess linker-payload stability, but it does not provide information on antibody-linker stability or the interplay between the two. Thus, in order to optimize the stability of an ADC, a screening assay is needed that can assess overall ADC plasma stability, by simultaneously deconvoluting the stability of both the linker and payload.9,12−14 Currently, several immunocapture and high resolution intactmass-analysis-based methodologies have been developed to characterize the drug to antibody ratio of ADCs as well as other ADC related species (e.g., catabolites) in biological samples.6,15−18 For cysteine−maleimide-based ADCs in particular, Wei et al. reported in 2016 a quantitative method, in which an antipayload antibody was used to extract and quantify all payload-associated antibodies and protein adducts in both in vitro and in vivo plasma samples.10 They were able to evaluate the species dependent formation of the albumin payload adducts with the method. However, in practice, an antipayload antibody is not always available, especially at the discovery screening stage when stability assays were conducted. In this report, we developed a novel two-step immunocapture approach to isolate the ADC and the plasma albumin payload adduct for respective quantification of the conjugated payloads using the protease specific for the linker used in the ADC conjugation. In addition, a more efficient cysteine protease, 19 papain, and a microwave-assisted digestion approach were evaluated and employed for a faster payload release assay, which was exemplified using an ADC (TAK-001) with a Takeda proprietary payload and a protease-cleavable dipeptide linker similar to an ADC drug TAK-264 (previously MLN0264), which is under clinical investigation.20

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MATERIALS AND METHODS

Chemicals, Reagents, and Apparatus. The test articles, Takeda proprietary payload (TPP) and TAK-001 ADC, were provided by the Drug Source at Takeda (Cambridge, MA). DLdithiothreitol (DTT), trifluoroacetic acid (TFA), sodium acetate (NaOAc), Tween 20, L-cysteine, cathepsin B from human liver, papain, and PureProteome Albumin Magnetic Beads were purchased from Millipore Sigma (St. Louis, MO or Milwaukee, IL). d8-Monomethyl auristatin E (d8-MMAE) was acquired from Albany Molecular Research Inc. (Albany, NY). CD-1 mouse and human K2EDTA plasma was purchased from Bioreclamation, LLC (Hicksville, NY). Acetonitrile, water, and formic acid (FA), which were HPLC grade or better, were purchased along with EZ-Link Sulfo-NHS-Biotin and Dynabeads M-280 (streptavidin coated magnetic beads) from Thermo Fisher (Chicago, IL). Stable isotope labeled human IgG1 heavy chain (SIL-HC) surrogate peptide was synthesized by Elim Biopharmaceuticals Inc. (Hayward, CA). Mass spectrometry grade lysyl endopeptidase (Lys-C) was purchased from Wako Chemicals USA (Richmond, VA), and Rapigest was purchased from Waters (Milford, MA). The antigen of the ADC was supplied internally by the Protein Sciences at Takeda (Cambridge, MA) and biotinylated in house. EDTA disodium solution (0.5 M, pH 8), 1× PBS buffer (pH 7.4), and PBST (PBS with 0.01% Tween 20) were prepared in house. The Rapid Enzyme Digestion Systems (REDS) was purchased from Hudson Surface Technology, Inc. (Old Tappan, NJ). The KingFisher Flex magnetic beads processor was purchased from ThermoFisher Scientific (Waltham, MA). B

DOI: 10.1021/acs.analchem.8b00694 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry stability sample was transferred to a 96-well Thermo Kingfisher plate with 0.1 mg of target antigen coupled M-280 beads already added (suspended in 100 μL of PBST, pH 7.4). Samples and magnetic beads were mixed well and incubated for 40 min at room temperature. Magnetic beads were then collected in 400 μL of PBST in completion of the first ADC depletion cycle. Another 0.1 mg of target antigen coupled M280 beads (suspended in 100 μL of PBST, pH 7.4) was added to the remaining supernatant for the second ADC depletion cycle. Again, samples and magnetic beads were mixed well and incubated for 40 min at room temperature. Magnetic beads used for the second ADC depletion cycle were also collected for washing steps. The collected magnetic beads from both depletions were washed separately with 400 μL of PBST twice followed by PBS and 10% acetonitrile in sequence prior to the release of the beads into 100 μL of papain activation solution. After the ADCs were depleted, approximately 200 μL of the remaining supernatant in the sample plate was transferred to a microcentrifuge tube and was mixed with 400 μL of prewashed PureProteome Albumin Magnetic Beads (∼133 mg). The beads were incubated for 60 min with the albumin payload adducts containing supernatant with end-over-end rotation at room temperature. After that, all supernatants were transferred to a new microcentrifuge tube for protein precipitation by adding 5 volumes of acetonitrile with 0.1% formic acid (1 mL), followed by centrifugation at 10 000g for 10 min. The antialbumin magnetic beads remaining in the microcentrifuge tubes were washed 4 times in a similar manner as for the M-280 beads, except that a washing volume of 800 μL was used. All of the protein pellets as well as the magnetic beads resulting from the previous immunocapture steps were collected and digested with papain. In brief, all samples were mixed well with 100 μL of activation buffer followed by adding 20 μL of activated papain solution (10 mg/mL). The digestion was carried out on REDS at 37 °C for 45 min. Digested samples were immediately acidified by adding 10 μL of 20% TFA. An aliquot (160 μL) of 6.25 ng/mL d8-MMAE (prepared in 95% acetonitrile and 0.1% formic acid in water) was spiked to the sample. All samples were then vortexed for 1 min and centrifuged at 2100g for 10 min. Then, 1 μL of supernatant was injected into the LC/MS/ MS system for analysis. The LLOQ for conjugated TPP was 0.2 ng/mL.

Figure 1. In vitro plasma stability profiles of (a) free payload TPP (1 μM at 37 °C, n = 3); (b) total antibody; (c) conjugated payload and deconjugated payload in mouse plasma; (d) conjugated payload and deconjugated payload in human plasma. ((b−d) completed with 10 μg/mL at 37 °C, n = 2.) Data are the average, and error bars represent the standard deviations.

actually not an efficient enzyme in processing the ADC in a plasma matrix.10 In our lab, we found that in the presence of a plasma matrix, the linker cleavage rate by cathepsin B could drop considerably compared to that in a neat buffer solution. Previously, Xu et al. also observed a significant reduction of dipeptide linker cleavage efficiency by cathepsin B under microwave-assisted deconjugation conditions.20 Hence, in order to achieve reasonably high cleavage efficiency for TAK-001 as well as the potential albumin adducts, a greater amount of cathepsin B would be needed, which is unfortunately cost prohibitive especially for a sizable ADC screening process. Recently, a few laboratories including ours successfully used a more cost-effective surrogate cysteine protease found in papaya, papain, to achieve cleavage of ADC dipeptide linkers after a minimum of 2−4 h of incubation (no microwave treatment).21,22 To reduce assay time, we further explored the use of the microwave technology to speed up the papain cleavage reactiona time-limiting step for the assay. The experiment was carried out by incubating 1 μg of TAK-001 with cathepsin B and papain for up to 40 min in REDS (37 °C, 400 W). The method for cathepsin B-based TPP release was adopted from Xu et al.,20 while the condition for papain-based TPP release was adopted from Sanderson et al.,21 with some modifications as detailed in the Materials and Methods section. As shown in Figure 2a, a more complete release of TPP was achieved with papain than with cathepsin B. No significant difference was found between the two papain concentrations

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RESULTS AND DISCUSSION Free Payload Plasma Stability Assessment. In order to understand the impact of potential instability of the free payload on the ADC stability in plasma, the plasma stability of TPP alone was evaluated in both mouse and human plasma at 1 μM before establishing plasma stability for TAK-001. As shown in Figure 1a, the average TPP peak area from LC/MS/MS analysis dropped only 5% compared with that at T0 after 6 days of incubation at 37 °C in human plasma. In mouse plasma, however, a more pronounced change of the peak area response was observed. The payload response decreased by 12% in 3 days and 29% in 6 days. The results suggested that TPP was stable in human plasma for up to 6 days and moderately stable in mouse plasma for at least 3 days. Optimization of Cleavage Enzyme and Digestion Time for Payload Release. TAK-001 contains a proteasecleavable dipeptide linker. Based on previous work with TAK264, which contains a similar linker,20 cathepsin B is a natural choice for releasing the conjugated payload enzymatically for LC/MS/MS-based quantification. However, cathepsin B was

Figure 2. Optimization of enzyme amount and incubation time: (a) papain vs cathepsin B in releasing payload from TAK-001 (n = 2); (b) time course of papain digestion with mouse plasma containing 10 μg/ mL of TAK-001 (n = 2). Data are the average, and error bars represent the standard deviations. C

DOI: 10.1021/acs.analchem.8b00694 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry

incubation in human plasma, in which the conjugated payload level dropped to 74% of its initial concentration in 6 days. Only a minimal amount of the deconjugated payload (0.2%) was detected in human plasma on day 6 by LC/MS/MS. The lower stability of TAK-001(indicated by higher deconjugation rate) in mouse vs human plasma was likely due to the high carboxylesterase 1C activity in mouse as reported in the literature for those dipeptide linker containing ADCs.24−26 However, this did not explain the clear discrepancy between the conjugated payload disappearance and the deconjugated payload formation within each speciesapproximately 18% for mouse plasma or 26% for human plasma. In both mouse and human plasma incubations, the payload was shown to be relatively stable (Figure 1a), and yet, only ∼80% of the total payload could be recovered from the immunocaptured ADC (under mass balance conditions, the expected deconjugated payload levels were illustrated by dotted lines in Figure 1c,d). Quantitation of Payload Adducts with Plasma Albumin and Other Proteins. Because native cysteine residues and maleimide anchors were used to generate this ADC, we hypothesized that the conjugated payload can undergo thiol exchanges with albumin as reported in the literature,1,7−11 which could result in migration of some linkerpayload molecules to albumin. Any putative albumin adducts would not be extractable for detection using our original onestep immunocapture methodology. In order to test the hypothesis, a novel two-step immunocapture workflow was designed to measure the potential loss of conjugated payload in each fraction (Figure 3). Briefly, target antigen was used in the

tested (0.2 mg, 1× vs 0.8 mg, 4× total). Therefore, the condition of 0.2 mg (1×) of papain per 10 μL of plasma samples was selected for follow-up experiments to further optimize the digestion time in spiked mouse plasma samples. It is worth pointing out that the enhanced method was intended for the cleavage of both ADC and potential albumin adducts. Since the reference standard of albumin adducts is not available, direct optimization of albumin adduct digestion could not be carried out. Considering that the dipeptide linkage in ADC and albumin adducts is identical, it is plausible to assume that the same cleavage condition would work equally well for potential albumin adducts. To confirm whether papain could be partially deactivated over time under the microwave condition, as it appeared for cathepsin B,22 in the follow-up experiment, a second aliquot of 20 μL of fresh papain was added at 45 min (from the start of digestion) to those aliquots scheduled to be quenched at 60 and 75 min (incubation volume corrected due to the dilution from the second addition of papain). In this case, if digestion was not complete due to the deactivation of papain, the remaining conjugated TPP would be cleaved off by freshly added papain, therefore resulting in an increase of the TPP concentration. As shown in Figure 2b, no increase of TPP concentration was observed in the samples with additional fresh papain after 60 and 75 min of digestion, which indicated that a complete payload release was achieved already in approximately 45 min. Therefore, 0.2 mg of papain and 45 min of microwaveassisted incubation time were chosen as the standard conditions for subsequent ADC stability and thiol-exchange assessment. Compared with the long incubation time used in cathepsin B cleavage reported by Wei et al.10 and Liu et al.,23 the significantly shortened incubation time using papain allowed us to complete a full bioanalysis with the two-step immunocapture-based sample preparation within a day. Quantitation of Total Antibody, Conjugated Payload, and Deconjugated Payload. Initially, total antibody and conjugated payload concentrations on days 0, 1, 2, 3, and 6 were measured by discrete single-step immunocapture LC/ MS/MS assays as described in the Supporting Information. As the antibody carrying toxin was a humanized antibody, a generic capturing reagent like antihuman Fc would not work in human plasma samples due to the presence of endogenous immunoglobulins. Target antigen was therefore used in this one-step immunocapture method to enrich the total antibody from human and mouse plasma stability samples. As shown in Figure 1b, the total antibody demonstrated excellent stability when incubated with human and mouse plasma at 37 °C for up to 6 days. On day 6, the measured concentrations of the total antibody in human and mouse plasma samples were still within ±1% of the spiked concentration at T0. The conjugated and deconjugated payload concentrations were measured and plotted in Figures 1c (mouse) and 1d (human) as T0-normalized values. Therefore, the percentage values shown refer to the measured payload (both free and conjugated forms) relative to the total spiked payload at T0. The free payload concentration for the spiked ADC at T0 was almost negligible. However, a sharp decline of the conjugated payload (from 107 to 43.3%) as well as a rapid formation of the deconjugated payload (from 1.30 to 45.6%) was observed for mouse plasma after 24 h of incubation. Following 6 days of incubation in mouse plasma, only 17% of the conjugated payload remained, while the deconjugated payload level rose only up to 64.7%. Interestingly, this was not the case in the

Figure 3. Bioanalytical workflow for quantifying plasma albumin payload adducts by ADC depletion followed by anti-albumin magnetic bead enrichment.

first immunocapture step to deplete all ADC molecules. Next, the resulting supernatant was subjected to the second immunocapture process, which was designed to enrich plasma albumin as well as any plasma albumin payload adducts. During this step, an immobilized antihuman plasma albumin ligand with cross-reactivity to rodent plasma albumin was used. Finally, proteins remaining in the ADC and albumindepleted supernatants were precipitated. The beads and pellets (containing nonalbumin protein payload adducts) were then digested by papain in REDS prior to LC/MS/MS analysis. As shown in Table 1 and Figure S-1a, in the day 6 mouse plasma sample, the payload levels of TPP-associated albumin and pellet-associated linker-payload adducts were found to be 13.6 and 0.4 ng/mL, respectively. By accounting for the thiol exchange, it seems that the majority of the payload could be recovered from the day 6 mouse plasma stability sample (93.7 ng/mL recovered out of 108 ng/mL total). In human plasma, the TPP level of albumin- and pellet-associated linker-payload D

DOI: 10.1021/acs.analchem.8b00694 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry

Table 1. Summary of Measured Payload Concentrations in Each Fraction of Mouse and Human Plasma Stability Samples ID

T1 A B C D E T2 T3

fraction

initial total free + deconjugated conjugated (first immunocapture) conjugated (second immunocapture) albumin adducts unknown protein adducts remaining conjugated payload total recovered payload

mouse plasma (n = 2)

human plasma (n = 2)

payload conc. ± SD (ng/mL)

payload conc. ± SD (ng/mL)

T0 97.1a 1.30 ± 0.04 103 ± 1 2.2 ± 0.9 1.1 ± 0.0 BQLc 105d 108e

day 6 63 ± 1 16.5 ± 0.5 0.40 ± 0.04 13.6 ± 0.1 0.4 ± 0.3 16.9d 93.7e

T0 97.1a BQLb 105 ± 5 2.5 ± 0.7 2.1 ± 0.1 BQLc 108d 110e

day 6 0.19 ± 0.01 71.8 ± 0.2 2.1 ± 0.3 16.9 ± 0.1 1.4 ± 0.2 74.0d 92.5e

Initial total payload concentration was converted from spiked ADC concentration (10 μg/mL). bBelow quantitation limit (0.05 ng/mL). cBelow quantitation limit (0.2 ng/mL). dSum of conjugated payload (depleted ADC) fractions = B + C. eSum of all recovered payload fractions = A + B + C + D + E. a

was developed for in vitro plasma stability study of cysteine− maleimide-based ADCs, it can also be used for thiol-exchange assessment of these ADCs dosed in vivo.

adducts were found to be 16.9 and 1.4 ng/mL, respectively, at day 6 (see Table 1 and Figure S-1b), suggesting a large portion of the previously undetected payload was actually present as linker-payload adducts. In both species, it was demonstrated that greater than 97% of ADC was depleted in one round of immunocapture with the target antigen attached beads. After two rounds of immunocapture, the assumption was made that the majority of ADC molecules were depleted and that any other conjugated forms of payload left in the solution would primarily be adducts of plasma albumin or other unidentified proteins (95%, relative to the initial total payload dose) was achieved with the new assay strategy, no additional effort was made to characterize other payload adducts that might form with other thiol-containing constituents that exist in plasma. This work demonstrates the necessity of conducting multiple assays for plasma stability assessment for ADCs. For instance, if only the formation of the free payload was monitored by assuming that all deconjugated payload was converted to the free form, it could lead to a false conclusion that TAK-001 had good stability in human plasma. Since an antipayload antibody was not available in this early discovery project, the antialbumin beads were used to determine antibody-linker stability. Originally designed for abundant plasma albumin removal in plasma samples,27,28 the anti-albumin magnetic beads were applied to the experiment in an unconventional manner for the enrichment of plasma albumin and its adducts. Unlike the antipayload antibody capture method reported by Wei et al.,10 the beads specifically isolated albumin payload adducts, therefore simplifying the interpretation of the stability results. Per the manufacturer’s data, the anti-albumin beads could capture 98% of albumin from human plasma and serum and approximately 88% of albumin from mouse serum, when the suggested beads-to-sample volume ratio is used.29 In this study, the amount of anti-albumin beads used was scaled down to match the estimated amount of albumin in 10 μL of plasma sample as suggested. Nevertheless, the complete capture of the albumin from plasma is not necessary, because as long as majority of the albumin was captured, the results are sufficient to evaluate the extent of thiol exchange. Alternatively, if albumin was only partially captured, the captured albumin could be digested and quantified using the surrogate peptide approach.30 Then, the total adduct estimation could be made by applying a scaling factor to the measured adduct with the scaling factor being the ratio of the theoretical total level of albumin to the captured albumin. Finally, although the method

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CONCLUSIONS A novel method was developed to provide a more informative assessment of ADC in vitro incubation stability in plasma of multiple species. In this method, a two-step immunocapture approach was used to isolate the ADC and the plasma albumin payload adduct for respective quantification of the conjugated payload using the protease specific for the linker used in the ADC conjugation. In addition, a more efficient cysteine protease, papain, and a microwave-assisted digestion approach were evaluated and employed for a faster payload release. Collectively, the developed assay assessed the stability of TAK001 ADC and helped to address the discrepancy discovered during a cysteine−maleimide-based ADC plasma stability study. The method is readily applicable to the evaluation of stability of ADCs that use thiol maleimide conjugation chemistry.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00694. Supplemental Methods; Figure S-1: Recovered payload concentration in each fraction of the plasma stability samples (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (L.D.) ORCID

Linlin Dong: 0000-0002-7944-8582 Present Address †

Chao Li, Nuventra Pharma Sciences, 2525 Meridian Parkway, Durham, North Carolina 27713, United States Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Francois Soucy, Gang Li, Hong Myung Lee, Mario Girard, Tianlin Xu, Stepan Vyskocil, Scott Freeze, and Tzu-Tshin Wong from the Discovery Chemistry group at E

DOI: 10.1021/acs.analchem.8b00694 Anal. Chem. XXXX, XXX, XXX−XXX

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(24) Francisco, J. A.; Cerveny, C. G.; Meyer, D. L.; Mixan, B. J.; Klussman, K.; Chace, D. F.; Rejniak, S. X.; Gordon, K. A.; DeBlanc, R.; Toki, B. E.; et al. Blood 2003, 102, 1458−1465. (25) Li, B.; Sedlacek, M.; Manoharan, I.; Boopathy, R.; Duysen, E. G.; Masson, P.; Lockridge, O. Biochem. Pharmacol. 2005, 70, 1673− 1684. (26) Dorywalska, M.; Dushin, R.; Moine, L.; Farias, S. E.; Zhou, D.; Navaratnam, T.; Lui, V.; Hasa-Moreno, A.; Casas, M. G.; Tran, T.-T.; Delaria, K.; Liu, S.-H.; Foletti, D.; O’Donnell, C. J.; Pons, J.; Shelton, D. L.; Rajpal, A.; Strop, P. Mol. Cancer Ther. 2016, 15, 958−970. (27) Mei, N.; Seale, B.; Ng, A. H. C.; Wheeler, A. R.; Oleschuk, R. Anal. Chem. 2014, 86, 8466−8472. (28) Sobot, D.; Mura, S.; Yesylevskyy, S. O.; Dalbin, L.; Cayre, F.; Bort, G.; Mougin, J.; Desmaële, D.; Lepetre-Mouelhi, S.; Pieters, G.; Andreiuk, B.; Klymchenko, A. S.; Paul, J.-L.; Ramseyer, C.; Couvreur, P. Nat. Commun. 2017, 8, 15678. (29) Millipore Sigma. Search Results for PureProteome Albumin Magnetic Beads. https://www.emdmillipore.com/US/en/search/ LSKMAGL10. (30) Liu, G.; Zhao, Y.; Angeles, A.; Hamuro, L. L.; Arnold, M. E.; Shen, J. X. Anal. Chem. 2014, 86, 8336−8343.

Takeda Boston for providing TAK-001 and TPP standards and Pamela Brauer, Jiejin Chen, and Qing Xu from the Protein Sciences group for providing the target antigen and related support. The authors also want to thank Ling Xu, Hiroshi Sugimoto, Kojo Abdul-Hadi, Debra Liao, Jean-Pierre Minembe, and Yinling Li from the DMPK group for their support and helpful discussions.

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DOI: 10.1021/acs.analchem.8b00694 Anal. Chem. XXXX, XXX, XXX−XXX