MS Bioanalysis of ProteinâDrug ConjugatesâThe Importance

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

LC-MS/MS bioanalysis of protein-drug conjugates - The importance of incorporating succinimide hydrolysis products Chuan Shi, Shalom Goldberg, Tricia Lin, Vadim Y. Dudkin, Wayne C. Widdison, Luke Harris, Sharon D. Wilhelm, Yazen Jmeian, Darryl Davis, Karyn O'Neil, Naidong Weng, and Wenying Jian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00411 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

LC-MS/MS bioanalysis of protein-drug conjugates -

The importance of incorporating succinimide hydrolysis products

Chuan Shi1,3, Shalom Goldberg1, Tricia Lin1, Vadim Dudkin1, Wayne Widdison2, Luke Harris2, Sharon Wilhelm2, Yazen Jmeian1, Darryl Davis1, Karyn O’Neil1, Naidong Weng1, Wenying Jian1* 1. Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477 2. ImmunoGen, Inc., 830 Winter Street, Waltham, MA 02451 3. Current affiliation: Wuhan Chuantai Technologies, 388 Gaoxinerlu, Wuhan, Hubei, China 430000

*Corresponding author: [email protected]

Key words: Antibody-drug conjugate (ADC), Protein-drug conjugate (PDC), Centyrin, Centyrin-drug Conjugate (CDC), Bioanalysis, LC-MS, maleimide, succinimide hydrolysis

ACS Paragon Plus Environment

1

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

Abstract Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure because it requires detection of both small and large molecules as one entity.

Existing

approaches to quantify the conjugated species of ADCs involve a ligand binding assay (LBA) for conjugated antibody or hybrid LBA/liquid chromatography-tandem mass spectrometry (LCMS/MS) for quantitation of conjugated drug. In our current work for a protein-drug conjugate (PDC) using the Centyrin scaffold, a similar concept to ADCs but with smaller protein size, an alternative method to quantify the conjugate by using a surrogate peptide approach was utilized. The His-tagged proteins were isolated from biological samples using immobilized metal affinity chromatography (IMAC), followed by trypsin digestion. The tryptic peptide containing the linker attached to the payload was used as a surrogate of the conjugate and monitored by LCMS/MS analysis. During method development and its application, we found that hydrolysis of the succinimide ring of the linker was ubiquitous, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in the animals, and ex vivo during the trypsin digestion step of the sample preparation. We have shown that hydrolysis during trypsin digestion is concentration-independent and consistent during the work flow therefore, having no impact on assay performance. However, for samples that have undergone extensive hydrolysis prior to trypsin digestion, significant bias could be introduced if only the non-hydrolyzed form is considered in the quantitation. Therefore, it is important to incorporate


2

Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


succinimide hydrolysis products in the quantitation method in order to provide an accurate estimation of the total conjugate level.

More importantly, the LC-MS/MS based method

described here provides a useful tool to quantitatively evaluate succinimide hydrolysis of ADCs in vivo, which has been previously reported to have significant impact on the stability, exposure, and efficacy.


3


Page 4 of 25

Introduction Antibody-drug conjugates (ADCs) have been increasingly recognized as important therapeutic agents for treatment of cancer, due to their targeted delivery of potent cytotoxic agents.

Four

ADCs, Mylotarg (gemtuzumab ozogamicin, subsequently withdrawn in 2010)1, Adcetris® (brentuximab vedotin)2, Kadcyla® (ado-trastuzumab emtansine)3, and Besponsa® (inotuzumab ozogamicin)4 have been approved by the US FDA and more are in the discovery and development pipelines of the pharmaceutical industry. In order to provide relevant information on drug exposure and safety/efficacy of ADCs, different species generated in vivo from ADCs have to be analyzed, which involves bioanalytical approaches for measurement of both large and small molecules. In general, the most common analytes for evaluation of pharmacokinetics (PK) of ADCs include total antibody measured by ligand binding assay (LBA), unconjugated small molecule payload by LC-MS assay, and conjugated antibody measured by LBA with use of antidrug antibody

5-7

. Recently, release of drug from the conjugate has been used as an alternative

measurement of the conjugated antibody, quantification of which involves affinity capture of the conjugate, cleavage of the linker, and LC-MS/MS analysis of the released small molecule payload

5,6,8,9

. In addition, methods have been developed to characterize relative abundance of

individual ADC species with different drug-to-antibody ratio (DAR) from in vivo samples by using affinity capture-LC-high resolution MS (HRMS)

10-13

. A novel approach using the DAR

profiles determined by LC-HRMS and total antibody measured by LBA to calculate concentrations of conjugated drug has also been recently proposed and applied in PK assessment of ADC in animals 14. Linker chemistry plays a crucial role in ADC efficacy and toxicity 15,16. In order to maximize the therapeutic window, particularly for ADCs containing cleavable linkers, the linker should be


4

Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


stable in systemic circulation while being easily cleaved to release the payload once the ADC is internalized into the target cancer cells.

Various linker chemistries, including hydrazones,

disulfides, thioethers, and peptides, have been explored

16-22

.

Among them, a thiosuccinimide

linkage, formed through the reaction of the thiol on a cysteine residue with a maleimide group has been widely used (Figure 1A). One of the reasons for its popularity is that Michael addition of thiols to maleimides is rapid under aqueous conditions and can provide nearly quantitative yields of conjugates without the need for a large excess of either species

23

.

However,

thiosuccinimide formation is potentially chemically reversible depending on the pKa of the conjugated thiol24,25, leading to slow release of the linked payload during prolonged circulation. The resulting maleimide group can react with thiol-containing components in a biological system such as cysteine, glutathione, or serum albumin (Figure 1A) 12,26. On the other hand, it has been shown that hydrolysis of the five-membered succinimide ring can greatly alter the stability of the thioether link

27,28

. Once the ring is opened through hydrolysis, the structure cannot undergo

retro-Michael reaction and subsequent thiol exchange (Figure 1A). Systematic studies have been conducted to elucidate the effects of succinimide ring opening on stability and therapeutic activities of ADCs 29-31. The results have shown that the ring-opened ADCs exhibit equivalent in vitro potency, and improved in vitro stability, in vivo exposure, and efficacy in comparison to their non-hydrolyzed counterparts

31

. Several efforts have been made to develop strategies to

promote the ring-open reaction in ADC products, such as modulation of the adjacent amino acids at the conjugate site to create a positively charged environment

30

, modification of chemical

structure on the linker to incorporate a basic amino group 29, a catalyst-promoted reaction 31,32, or incorporation of electron-withdrawing N-substituents on the succinimide ring 33.


5


Page 6 of 25

In recent years, alternative protein scaffolds that share properties of antibodies in terms of their specificity and potency but with smaller size and simpler structures 34,35 have been used in place of antibodies to develop novel conjugated therapeutic agents 36,37. Similar to ADCs, protein-drug conjugates (PDCs) consist of a protein targeting agent and payload, conjugated through a linker. They have a smaller and simpler structure than ADCs and typically feature site-specific conjugation, while the linker and payload chemistry from the current ADC platforms can be retained. We are developing PDCs using the Centyrin scaffold, namely Centyrin-drug conjugate (CDC), an alternative scaffold based on the consensus sequence of fibronectin type III domains (FN3 domains) from human Tenascin C 38. In our recent effort to develop PDCs with the Centyrin scaffold, the need to quantify conjugates in in vitro and in vivo biological samples has arisen.

Due to lack of specific antibodies for the

payload and the protein, and uncleavable nature of the linkers of PDCs investigated in this study, instead of using LBA to measure the protein conjugates or hybrid LBA/LC-MS/MS to measure drug conjugated to protein, an LC-MS/MS approach was used. In this approach, tryptic peptides of the protein carrier that contain the thiosuccinimide linkers and the payload are measured, as a surrogate of the conjugate, using LC-MS/MS. During this work, we have found extensive hydrolysis of the succinimide ring on the linker, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in animals, and ex vivo during trypsin digestion. Therefore, it is important to incorporate the hydrolyzed forms in the MS method in order to accurately evaluate in vitro stability and in vivo exposure of the PDC. This approach can provide the capability to investigate individual succinimide hydrolysis forms in biological samples. Experimental Section


6

Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Materials: Protein-drug conjugate EEB9 was prepared by ImmunoGen, Inc., and the stable isotopic labeled (SIL) protein internal standard (IS, generated by E. coli protein expression system with 13C6, 15N-Leu) were obtained internally at Janssen Research & Development, LLC. N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) treated trypsin from bovine pancreas was purchased from Sigma-Aldrich (St. Louis, MO, USA). Immobilized metal affinity chromatography (IMAC) 96-well plates (HisPur Ni-NTA spin plates) were purchased from Thermo Scientific (Rockford, IL, USA). Blank heparin mouse plasma was purchased from Bioreclamation (Hicksville, NY). Quantitative analysis and preparation of plasma standards and quality control (QC) samples: For absolute quantitation of EEB9, calibration curve and QC samples were prepared by serial dilution of PDC stock solutions (33 µM) with blank mouse plasma. PDC calibration curve concentrations were 6.6, 16.5, 33, 165, 330, 1651, 3302, 4954, and 6606 nM, while QC samples were prepared at concentrations of 10, 99, 661, and 5285 nM. The calibration curve was established from peak area ratio of non-hydrolyzed analyte (0 water)/IS without incorporating the hydrolyzed form, or the sum of both non-hydrolyzed and hydrolyzed forms/IS [(0 water+1 water+2 water)/IS] using a linear regression with a weighting of 1/x2. Sample preparation: The IMAC plate was conditioned with 250 µL of wash buffer (10 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole) three times. Plasma sample (50 µL) was mixed with 10 µL of SIL protein IS (100 µg/mL in 10% (v/v) acetonitrile:water) and 100 µL of wash buffer. The mixture was loaded onto the IMAC plate and incubated for 30 minutes at room temperature on a plate vortexer at 700 RPM. The samples were then washed three times with 250 µL of wash buffer and eluted with 100 µL of ammonium acetate buffer (10 mM, pH 3.8) into a


7


Page 8 of 25

96-well polypropylene plate. 20 µL of 500 mM ammonium bicarbonate buffer and 20 µL trypsin (1 mg/mL in 50 mM acetic acid) were added to the eluent (final pH 8.2). The plate was incubated at 37 ºC for 4 hours and reaction was quenched with 10 µL of 10% trifluoroacetic acid (TFA). The final solution (10 µL) was injected for LC-MS analysis. LC-MS conditions: Both LC-HRMS and LC-multiple reaction monitoring (MRM) analysis employed a HPLC system consisting of Shimadzu LC20AD pumps and a SIL-HTC autosampler (Columbia, MD). A Kinetex XB-C18 column (2.1 mm × 50 mm, 2.6 µm, 100 Å, Phenomenex, Torrance, CA) was used for LC separation at a flow rate of 0.4 mL/min. Mobile phase A was 0.2% formic acid (FA) in water, and mobile phase B was 0.2% FA in acetonitrile. The needle rinse solvent was 0.1% TFA and 50% acetonitrile in water (v/v/v). A linear gradient of 5-90% B was used and the run time was 6 min for LC-HRMS analysis and 5 min for LC-MRM analysis. LC-HRMS analysis was conducted on an API 5600 triple TOF (Q-TOF) mass spectrometer (AB Sciex, Foster City, CA) by information dependent acquisition (IDA) to trigger product ion spectrum acquisition by intensity. The multiple reaction monitoring (MRM) analysis was carried out with an API 5000 triple-quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). The MRM transitions were 1167.9 > 485.2 (EEB9, 0 water, 3+), 1173.9 > 485.2 (EEB9, +1 water, 3+), 1179.9 > 485.2 (EEB9, +2 water, 3+), and 825.1 > 965.6 (IS, 3+). Evaluation of succinimide hydrolysis during trypsin digestion: PDC stock solution (33 µM) was diluted to 1.65 µM with blank mouse plasma. Three aliquots of 50 µL each were purified by IMAC plates and the pH of the eluates were adjusted to 6.4, 7.1, and 8.2 respectively with ammonium hydroxide solution. Ammonium bicarbonate buffer (20 µL of 500 mM) and 20 µL of trypsin solution (1 mg/mL in 50 mM ammonium bicarbonate buffer) were added to the eluent


8

Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and incubated at 37 ºC. The final mixture (10 µL) was injected for LC-MRM analysis after incubating for 2 h, 4 h, 8 h, or 20 h. In vitro plasma stability: EEB9 stock solution (33 µM) was spiked into blank mouse plasma at a concentration of 3.3 µM and aliquotted into 150 µL portions in individual microcentrifuge tubes. The tubes were incubated at 37 ºC in a water bath. At each time point (0 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, and 7 days), one tube was removed from the water bath and stored at -20 ºC until sample processing and analysis. Mouse PK study: EEB9 was intravenously (IV) administered to 9 Balb/c mice at 10 mg/kg (Centyrin dose). At each time point post-treatment (6 h, 24 h, and 48 h), three mice were euthanized and plasma samples were collected and stored at -80 ºC until sample processing and analysis. Results and Discussion Analytical Strategy Recent developments in analytical technologies allow the detection of large molecules using LCMS with excellent specificity and selectivity 39. A typical workflow to quantify large molecules involves enzymatic digestion of the molecule and measurement of a selected peptide as a surrogate using LC-MS/MS 39,40. In the current work, a surrogate peptide LC-MS/MS approach to quantify the PDC conjugates in biological samples was used. antibodies, instead of using immune-affinity capture,

Due to lack of specific

immobilized metal affinity

chromatography (IMAC) resin was used to isolate the conjugates from complex biological samples. The isolated proteins were then subjected to trypsin digestion. The particular tryptic peptide that contains the cysteine residue and the conjugated payload was measured using an LC-


9


Page 10 of 25

MRM method as a surrogate of the conjugate. Also, by selecting an additional surrogate peptide that does not contain the conjugation site, total protein (or total antibody in the case of an ADC) level can be quantified in the same assay. During the method development of this surrogate peptide assay, we found that succinimide ring hydrolysis occurred readily during multiple stages in the lifetime of the PDC.

Thus the assay

set-up, i.e. the MRM method, needs to account for both hydrolyzed and non-hydrolyzed forms. This may not be a significant issue if the extent of hydrolysis is consistent between the sample and reference standard, as shown in the example of quantifying spiked QC samples in the following section.

However, when there is a much higher extent of hydrolysis in the test

samples than the reference standard, a large proportion of the analytes will be missed in the MS detection if the MRM transitions only include the non-hydrolyzed form. Another advantage of including the MRM transitions of the hydrolysis form is to provide a tool to monitor the dynamics of each individual form, which are believed to possess different stability and efficacy properties. Identification of Succinimide Hydrolysis Products To set up the surrogate peptide LC-MRM method, we conducted trypsin digestion of the neat reference material, followed by MS-triggered MS2 analysis on HRMS to identify the suitable molecule ions and product ions for MRM transition.

The neat PDC reference standard was

digested with trypsin overnight and the resulting sample was subjected to LC separation, followed by analysis on a QTOF instrument using Information Dependent Acquisition (IDA), in which the HRMS full scan signal above a certain threshold triggers a product ion scan. Figure 2 shows the results for EEB9, which is a 258 amino acid protein containing two identical Centyrin motifs, each having a cysteine residue conjugated to the maytansinoid tubulin inhibitor DM1


10

Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


through a tri-glycine containing peptide linker CX1 21 (Figure 1B and 1C). Figure 2A shows the total ion current (TIC) of the high-resolution TOF MS scan. By extracting the theoretical m/z from the TIC, it was found that the payload-linker containing peptide eluted at around 3.3 min. Figure 2B shows the magnified HRMS spectrum (m/z 1160-1200) of the selected time span in Figure 2A. The spectrum shows two more species in addition to the peaks corresponding to the theoretical m/z of payload-linker containing peptide (m/z 1167.8515, charge state of 3), and they are tentatively assigned as the hydrolysis products with one additional water (m/z 1173.8575, charge state of 3) and two additional water molecules (m/z 1179.8637, charge state of 3), respectively. The extracted ion chromatography (XIC) of each species using an extraction window of 100 mDa gives distinctive peaks and the hydrolysis products eluted slightly earlier due to the increased polarity after addition of water (Figure 2C). The MS2 fragmentation of each species (Figure 2D) was similar and the characteristic fragments of the payload DM1 (m/z 453, 485, 547) can be found in all the species. In addition, y ions and b ions of the peptides that matched theoretical m/z within 10 ppm error were also consistent in each species. Because fragments of payload (m/z 453, 485, 547) and peptide (y ions and b ions) in the hydrolyzed forms were same as those in the non-hydrolyzed form and there are two succinimide rings on the linker, it is reasonable to believe that the +18 Da and +36 Da species correspond to the hydrolysis products of one and two succinimide rings on the linker, respectively (Figure 1D). Since water can attack either carbon of the imide group (Figure 1A) on either one or both of succinimide rings (Figure 1D), each hydrolyzed species presumably consisted of multiple isoforms which were not resolved in our short LC run. Hydrolysis during Trypsin Digestion and Evaluation of its Impact on Assay Performance


11


Page 12 of 25

To quantify the payload-linker containing peptide derived from trypsin digestion of PDC, an MRM method with transitions of the non-hydrolyzed and hydrolyzed forms was established and optimized based on the molecular ions and product ions observed in the IDA experiment. The MRM method monitored three different forms of the payload-linker containing peptide: (1) +0 water, the one on which both succinimide rings remained non-hydrolyzed; (2) +1 water, the one on which one of the succinimide rings has been hydrolyzed; (3) +2 water, the one on which both succinimide rings have been hydrolyzed. An experiment was conducted to evaluate the dynamics of succinimide hydrolysis during the trypsin digestion step of the sample preparation, which involves basic pH incubation - a condition facilitating succinimide ring hydrolysis. The signal intensity of each form (+0 water, +1 water, +2 water) was monitored during 20 hours of trypsin digestion at 37 °C (Figure 3A) of the neat EEB9 reference standard. At an early stage of digestion (0-4 h), the MS signal of all three forms increased due to digestion that generated additional tryptic peptides as well as the ongoing hydrolysis of the succinimide on the linker. As digestion progressed, there was a continued increase in +2 water form with a concomitant drop in the signal of +0 water and the +1 water forms. This was speculated to be due to on-going hydrolysis reaction that converted +0 and +1 water forms to +2 water form. The sum of signal intensity of all three forms, reflecting the total level of peptides generated during digestion regardless of hydrolysis status, decreased upon prolonged digestion (after 4h), most likely as a result of non-specific digestion that degraded the initially generated peptides, a phenomena often observed when low-grade trypsin is used in the experiments.

To avoid the non-specific

digestion, the final method was limited to 4 h of incubation before the reaction was quenched with acid.


12

Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Because succinimide hydrolysis during trypsin digestion might introduce complexity and postsampling artifacts, the digestion process was tested at lower pH in an effort to eliminate the hydrolysis reaction (Figure 3B). The extent of succinimide hydrolysis was evaluated by a relative quantitation approach using the peak area ratio of hydrolyzed peptides (sum of +1 water and +2 water forms) and the +0 water form. In a 20 h digestion, lower pH (pH 6.4 and pH 7.1) slightly reduced the extent of hydrolysis, which is consistent with the report that hydrolysis of the succinimide was found to be catalyzed by base

41

. Eluents in different buffers such as formic

acid or imidazole followed by pH adjustment by ammonium bicarbonate were also evaluated. However, succinimide hydrolysis appeared to be inevitable and it was not remarkably impacted by pH or buffer system.

In addition, in LC-HRMS analysis of the neat reference materials

without digestion, hydrolyzed species were also observed in the deconvoluted MS intact spectrum (Supporting Information), indicating that succinimide ring hydrolysis may already have taken place. Therefore, rather than eliminate hydrolysis, which does not appear to be possible, a better approach is to incorporate hydrolysis in the MS detection. The LC-MRM assay was then applied to analysis of PDCs in biological samples. EEB9 was spiked into mouse plasma at concentrations ranging from 6.6 nM to 6606 nM. The conjugate was captured by IMAC resin, and subjected to trypsin digestion for 4 h at 37 °C at pH 8.2. The resulting tryptic peptides were analyzed using the MRM method mentioned above to monitor both non-hydrolyzed (+0 water) and hydrolyzed forms (+1 water, +2 water). QC samples (n=3) prepared at 4 different levels of EEB9 were found to have similar extents of hydrolysis as indicated by the peak area ratio of hydrolyzed (sum of +1 water and +2 water) and nonhydrolyzed (+0 water) peptides of about 1.4 for all the samples (Figure 4). Consequently, hydrolysis of succinimide linker during trypsin digestion was expected not to introduce variation


13


Page 14 of 25

into the quantitation assay. This is confirmed by comparing the assay performance with or without incorporation of the hydrolysis products (Table 1). The concentration of conjugates was calculated using the peak area of +0 water form only, or the sum of the three forms (+0 water, +1 water, +2 water), and the calculated assay performance indicates that incorporation of succinimide hydrolysis products had minimal impact on accuracy and precision. Examination of Succinimide Hydrolysis prior to Sample Preparation Hydrolysis of the succinimide component of the linker of the ADC during circulation in vivo has been observed previously using qualitative methods

30

. The most important advantage of the

current approach of a surrogate peptide LC-MS/MS assay with incorporation of hydrolysis products is that it allows us to quantitatively monitor the hydrolysis of ADC/PDC linker. As a result, the investigation of overall ADC/PDC stability and efficacy can be more thorough because the assay can quantify the distinct hydrolyzed forms, which may have considerably different stability and efficacy. In an effort to evaluate plasma stability of the PDC, we conducted an in vitro incubation. EEB9 was spiked into mouse plasma, incubated at 37 ºC over a period of 7 days, and processed using IMAC and trypsin digestion followed by LC-MRM analysis. As shown in Figure 5A, the level of hydrolysis forms (+1 water, +2 water), expressed as the peak area, increased during the first 24 h of incubation while the non-hydrolyzed form (+0 water) dropped rapidly. In the following 6 days, the extent of hydrolysis increased significantly. The peak area ratio of hydrolyzed (sum of +1 water and +2 water) to non-hydrolyzed (+0 water) forms, as shown in Figure 5B, increased significantly from 1.5 to 79. When the reference standard that contains a hydrolyzed/nonhydrolyzed forms at a ratio of ~1.4 (the initial ratio) was used to conduct quantitation, extensive hydrolysis in the incubation samples resulted in a considerable difference in the plasma stability


14

Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


profile, with and without considering the hydrolysis products (Figure 5C). When the quantitation was solely based on the non-hydrolyzed form (+0 water), the time-concentration curves showed relatively rapid loss of the conjugate, while a much slower clearance is obtained by quantitation based on sum of all three forms (+0 water, +1 water, and +2 water). If the hydrolysis products were not incorporated in the quantitation method, significant underestimation of plasma stability could result. Applications of the Assay for in vivo Samples The assay was applied to PK evaluation of PDCs in mice. Mice were intravenously dosed with EEB9 and euthanized at various time points. Plasma samples were collected and analyzed using the IMAC-digestion-LC-MRM workflow as described above. All three forms, +0 water, +1 water, and +2 water, of the payload-linker containing peptides were monitored. As shown in Figure 6, there was a clear time-dependent trend where the ratio of hydrolyzed (sum of +1 water and +2 water) to non-hydrolyzed (+0 water) forms increased. Interestingly, the extent of hydrolysis in dosed animals was significantly lower than it was in the in vitro incubation of spiked plasma. At 48 h, the ratio of hydrolyzed to non-hydrolyzed forms was below 3 in the in vivo samples, while it was 7.4 in the in vitro plasma samples. Another observation is that the concentration of conjugate decreased much faster than that in the in vitro plasma incubation experiment, indicating additional catabolism pathways other than plasma proteolysis presumably target-mediated elimination, tissue-based proteolysis, and/or urinary clearance. The comparison of calculated plasma concentrations of the conjugates with and without incorporation of the hydrolysis forms are shown in Table 2. At the 6 h and 24 h time points, when hydrolysis was less extensive, the measured concentrations using non-hydrolyzed form only were not significantly different from those calculated based on the sum of non-hydrolyzed and hydrolyzed


15


Page 16 of 25

forms. At 48 h, the difference was relatively larger due to more extensive hydrolysis. However, due to the low absolute concentrations at this time point, missing the hydrolysis form would not make a significant impact on the overall PK parameters. If a PDC with longer half-life is evaluated, the importance of incorporating the hydrolysis forms would be greater.

It was also

observed that the extent of hydrolysis was different for PDCs with different linker structures. This suggests that the hydrolysis of succinimide linker is structure dependent, and therefore a method that can quantify both non-hydrolyzed and hydrolyzed forms will be very important for accurate PK evaluation of different ADC/PDCs. Conclusion A surrogate peptide LC-MS/MS bioanalytical method was used to quantify PDCs in mouse plasma for both in vitro and in vivo samples. The plasma samples were processed using IMAC resin and trypsin digestion, followed by LC-MRM analysis. The peptide containing the linker and payload was used as the surrogate for the conjugated species of the PDC. It was found that the succinimide rings on the linker underwent hydrolysis during trypsin digestion or prior to sample preparation. While hydrolysis during trypsin digestion has been shown to have no impact on assay performance, it is important to incorporate the hydrolyzed forms in the quantitation. Without adding the hydrolyzed forms in the quantitation method, significant bias could be introduced for samples that have undergone extensive hydrolysis prior to trypsin digestion. The current LC-MS/MS based method provides a useful tool to quantitatively evaluate different succinimide hydrolysis forms of ADC/PDC in vivo.

Previous studies have shown that

succinimide hydrolysis could introduce significant improvements in the stability, exposure, and efficacy of the ADCs

29-31

. Therefore, it is important to develop bioanalytical method that can


16

Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


monitor the hydrolysis. Prior to the current work, there had not been effective quantitation approaches to track hydrolysis in biological samples. The LBA-based conjugate assays do not have the capability to differentiate the subtle change in the succinimide rings.

The hybrid

LBA/LC-MS/MS based assay for drug conjugate requires release of the free drug via chemical or enzymatic reaction and therefore may lose the information on the linker. MS analysis of intact ADCs/PDCs does not have the resolving power to unambiguously and quantitatively differentiate the different hydrolysis forms at different sites. The current surrogate peptide approach, on the other hand, takes advantage of the specificity provided by MS at the peptide level and affords the capability to monitor succinimide ring hydrolysis as one of the important transformations of ADCs/PDCs.

Supporting Information Experimental description of intact LC-HRMS analysis of the neat solution of EEB9 and deconvoluted intact mass spectrum showing the non-hydrolyzed and hydrolyzed forms of EEB9 molecule. Acknowledgements The authors gratefully acknowledge ImmunoGen, Inc. for assistance in the design and synthesis of the test articles. The authors thank Dr. Thomas A. Keating, Dr Ravi V.J. Chari, Dr. Nathan Fishkin, and Dr. Alexis Moran for valuable review and comments of the manuscript. References (1)

Bross, P.F.; Beitz, J.; Chen, G.; Chen, X.H.; Duffy, E.; Kieffer, L.; Roy, S.; Sridhara, R.; Rahman, A.; Williams, G.; Pazdur, R. Clin Cancer Res 2001, 7, 1490.


17


(2)

(3) (4) (5) (6) (7) (8)

(9) (10) (11) (12) (13) (14) (15) (16) (17)

(18) (19)

(20)

(21)

(22)

Page 18 of 25

de Claro, R.A.; McGinn, K.; Kwitkowski, V.; Bullock, J.; Khandelwal, A.; Habtemariam, B.; Ouyang, Y.; Saber, H.; Lee, K.; Koti, K.; Rothmann, M.; Shapiro, M.; Borrego, F.; Clouse, K.; Chen, X.H.; Brown, J.; Akinsanya, L.; Kane, R.; Kaminskas, E.; Farrell, A.; Pazdur, R. Clin Cancer Res 2012, 18, 5845. Krop, I.; Winer, E.P. Clin Cancer Res 2014, 20, 15. Lamb, Y.N. Drugs 2017, 77, 1603. Gorovits, B.; Alley, S.C.; Bilic, S.; Booth, B.; Kaur, S.; Oldfield, P.; Purushothama, S.; Rao, C.; Shord, S.; Siguenza, P. Bioanalysis 2013, 5, 997. Kaur, S.; Xu, K.; Saad, O.M.; Dere, R.C.; Carrasco-Triguero, M. Bioanalysis 2013, 5, 201. Stephan, J.P.; Kozak, K.R.; Wong, W.L. Bioanalysis 2011, 3, 677. 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, 54. Rago, B.; Tumey, L.N.; Wei, C.; Barletta, F.; Clark, T.; Hansel, S.; Han, X. Bioconjug Chem 2017, 28, 620. Xu, K.; Liu, L.; Dere, R.; Mai, E.; Erickson, R.; Hendricks, A.; Lin, K.; Junutula, J.R.; Kaur, S. Bioanalysis 2013, 5, 1057. 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. Wei, C.; Zhang, G.; Clark, T.; Barletta, F.; Tumey, L.N.; Rago, B.; Hansel, S.; Han, X. Anal Chem 2016, 88, 4979. He, J.; Su, D.; Ng, C.; Liu, L.; Yu, S.F.; Pillow, T.H.; Del Rosario, G.; Darwish, M.; Lee, B.C.; Ohri, R.; Zhou, H.; Wang, X.; Lu, J.; Kaur, S.; Xu, K. Anal Chem 2017, 89, 5476. 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. Alley, S.C.; Benjamin, D.R.; Jeffrey, S.C.; Okeley, N.M.; Meyer, D.L.; Sanderson, R.J.; Senter, P.D. Bioconjug Chem 2008, 19, 759. Ducry, L.; Stump, B. Bioconjug Chem 2010, 21, 5. Boghaert, E.R.; Khandke, K.M.; Sridharan, L.; Dougher, M.; DiJoseph, J.F.; Kunz, A.; Hamann, P.R.; Moran, J.; Chaudhary, I.; Damle, N.K. Cancer Chemother Pharmacol 2008, 61, 1027. Xie, H.; Audette, C.; Hoffee, M.; Lambert, J.M.; Blattler, W.A. J Pharmacol Exp Ther 2004, 308, 1073. Hamann, P.R.; Hinman, L.M.; Hollander, I.; Beyer, C.F.; Lindh, D.; Holcomb, R.; Hallett, W.; Tsou, H.R.; Upeslacis, J.; Shochat, D.; Mountain, A.; Flowers, D.A.; Bernstein, I. Bioconjug Chem 2002, 13, 47. Sutherland, M.S.; Sanderson, R.J.; Gordon, K.A.; Andreyka, J.; Cerveny, C.G.; Yu, C.; Lewis, T.S.; Meyer, D.L.; Zabinski, R.F.; Doronina, S.O.; Senter, P.D.; Law, C.L.; Wahl, A.F. J Biol Chem 2006, 281, 10540. Singh, R.; Setiady, Y.Y.; Ponte, J.; Kovtun, Y.V.; Lai, K.C.; Hong, E.E.; Fishkin, N.; Dong, L.; Jones, G.E.; Coccia, J.A.; Lanieri, L.; Veale, K.; Costoplus, J.A.; Skaletskaya, A.; Gabriel, R.; Salomon, P.; Wu, R.; Qiu, Q.; Erickson, H.K.; Lambert, J.M.; Chari, R.V.; Widdison, W.C. Mol Cancer Ther 2016, 15, 1311. Sadowsky, J.D.; Pillow, T.H.; Chen, J.; Fan, F.; He, C.; Wang, Y.; Yan, G.; Yao, H.; Xu, Z.; Martin, S.; Zhang, D.; Chu, P.; Dela Cruz-Chuh, J.; O'Donohue, A.; Li, G.; Del


18

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) (24) (25) (26) (27) (28) (29)

(30)

(31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41)

Rosario, G.; He, J.; Liu, L.; Ng, C.; Su, D.; Lewis Phillips, G.D.; Kozak, K.R.; Yu, S.F.; Xu, K.; Leipold, D.; Wai, J. Bioconjug Chem 2017, 28, 2086. Chari, R.V.; Miller, M.L.; Widdison, W.C. Angew Chem Int Ed Engl 2014, 53, 3796. Baldwin, A.D.; Kiick, K.L. Bioconjug Chem 2011, 22, 1946. Ryan, C.P.; Smith, M.E.; Schumacher, F.F.; Grohmann, D.; Papaioannou, D.; Waksman, G.; Werner, F.; Baker, J.R.; Caddick, S. Chem Commun (Camb) 2011, 47, 5452. Lin, D.; Saleh, S.; Liebler, D.C. Chem Res Toxicol 2008, 21, 2361. Khan, M.N. J Pharm Sci 1984, 73, 1767. Knight, P. Biochem J 1979, 179, 191. Lyon, R.P.; Setter, J.R.; Bovee, T.D.; Doronina, S.O.; Hunter, J.H.; Anderson, M.E.; Balasubramanian, C.L.; Duniho, S.M.; Leiske, C.I.; Li, F.; Senter, P.D. Nat Biotechnol 2014, 32, 1059. Shen, B.Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K.L.; Tien, J.; Yu, S.F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O.M.; Scales, S.J.; McDonald, P.J.; Hass, P.E.; Eigenbrot, C.; Nguyen, T.; Solis, W.A.; Fuji, R.N.; Flagella, K.M.; Patel, D.; Spencer, S.D.; Khawli, L.A.; Ebens, A.; Wong, W.L.; Vandlen, R.; Kaur, S.; Sliwkowski, M.X.; Scheller, R.H.; Polakis, P.; Junutula, J.R. Nat Biotechnol 2012, 30, 184. Tumey, L.N.; Charati, M.; He, T.; Sousa, E.; Ma, D.; Han, X.; Clark, T.; Casavant, J.; Loganzo, F.; Barletta, F.; Lucas, J.; Graziani, E.I. Bioconjug Chem 2014, 25, 1871. Kalia, J.; Raines, R.T. Bioorg Med Chem Lett 2007, 17, 6286. Fontaine, S.D.; Reid, R.; Robinson, L.; Ashley, G.W.; Santi, D.V. Bioconjug Chem 2015, 26, 145. Binz, H.K.; Pluckthun, A. Curr Opin Biotechnol 2005, 16, 459. Jost, C.; Pluckthun, A. Curr Opin Struct Biol 2014, 27, 102. Simon, M.; Frey, R.; Zangemeister-Wittke, U.; Pluckthun, A. Bioconjug Chem 2013, 24, 1955. Goldberg, S.D.; Cardoso, R.M.; Lin, T.; Spinka-Doms, T.; Klein, D.; Jacobs, S.A.; Dudkin, V.; Gilliland, G.; O'Neil, K.T. Protein Eng Des Sel 2016, 29, 563. Jacobs, S.A.; Diem, M.D.; Luo, J.; Teplyakov, A.; Obmolova, G.; Malia, T.; Gilliland, G.L.; O'Neil, K.T. Protein Eng Des Sel 2012, 25, 107. Zhang, W.; Jian, W. Reviews in Analytical Chemistry 2014, 33, 31. Li, F.; Fast, D.; Michael, S. Bioanalysis 2011, 3, 2459. Khan, M.N.A.A.K. The Journal of Organic Chemistry 1975, 40, 1793.


19


Page 20 of 25

Figure 1. (A) Hydrolysis scheme of succinimide ring; (B) Sequence of Centyrin-drug conjugate (CDC) EEB9. The sequence of surrogate peptide for CDC is shown in blue and the two cysteine residues for conjugation are highlighted in yellow. The surrogate peptide for total Centyrin is shown in red. (C) Structure of EEB9. It is consisted of two identical Centyrin motifs each containing one cysteine residue for conjugation, and an albumin binding domain. (D) Structure of linker and payload. The inserted boxes show the structure of the hydrolysis products of succinimide rings on the linker.


20

Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. LC-MS analysis of tryptic digestion products of neat reference standard of EEB9. (A) TIC of full HRMS scan; (B) Zoomed-in HRMS scan spectrum at retention time of 3.3 min; (C) Extracted ion current of the payload/linker containing peptide at m/z 1167.85 (non-hydrolyzed, +0 water, top), m/z 1173.86 (hydrolyzed, +1 water, middle), and m/z 1179.86 (hydrolyzed, +2 water, bottom) using extraction window of 100 mDa; (D) Product ion spectra of 1167.85 (nonhydrolyzed, +0 water, top), m/z 1173.86 (hydrolyzed, +1 water, middle), and m/z 1179.86 (hydrolyzed, +2 water, bottom).


21


Figure 3. Hydrolysis of succinimide ring of EEB9 during trypsin digestion. (A) Time course of peak area of the non-hydrolyzed form (+0 water), hydrolyzed forms (+1 water, +2 water) and their sum (total) of payload/linker containing peptide during trypsin digestion at pH 8.2, 37 °C; (B) Time course of peak area ratio of hydrolyzed (sum of +1 water and +2 water)/nonhydrolyzed (+0 water) forms of payload/linker containing peptide at different pH, 37 °C. B 3.0E+05

0water

2.5E+05

1water

Peak Area Ratio of Hydrolyzed/Non-hydrolyzed

A

2water

Peak Area

2.0E+05

total

1.5E+05 1.0E+05 5.0E+04 0.0E+00 0

5

10 15 Time (h)

20

25

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

pH6.4 pH7.1 pH8.2 0

5

10 15 Time (h)

20

25

Figure 4. Peak area ratio of hydrolyzed (sum of +1 water and +2 water) and non-hydrolyzed (+0 water) forms of payload/linker containing peptide of EEB9 in QC samples at different concentrations (n=3, ± standard error of the mean).


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

n=3

10

99 661 Concentration (nM)


5285

22

Page 23 of 25

0water

3.5E+06

1water 3.0E+06

2water sum

Peak Area

2.5E+06

C 120

B

100

Quantitation using non-hydrolyzed form only Quantitation using both hydrolyzed and non-hydrolyzed forms

80 60

100

40 20 0 0

100

2.0E+06

200

Time (h)

1.5E+06 1.0E+06

Concentration (µg/mL)

A


Figure 5. Succinimide hydrolysis of EEB9 in the in vitro mouse plasma stability evaluation. (A) Time course of peak area of the non-hydrolyzed form (+0 water), hydrolyzed forms (+1 water, +2 water) and their sum of payload/linker-containing peptide during in vitro plasma incubation; (B) Peak area ratio of hydrolyzed (sum of +1 water and +2 water) and non-hydrolyzed (+0 water) forms during in vitro plasma incubation; (C) In vitro plasma stability profile with and without incorporating the hydrolyzed forms.

80 60 40 20

5.0E+05

0

0.0E+00 0

50

100 Time (h)

150

200

0

50

100 Time (h)

150

200

Figure 6. Peak area ratio of hydrolyzed (sum of +1 water and +2 water) and non-hydrolyzed (+0 water) forms of payload/linker-containing peptide in mice dosed with EEB9 (n=3, ± standard error of the mean). 2.0 1.9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0

20

40 Time (h)

60


23


Page 24 of 25

Table 1. Accuracy and precision of conjugate concentration determination in EEB9 mouse plasma QC samples without and with incorporation of the succinimide linker hydrolysis forms. Use sum of non-hydrolyzed and hydrolyzed forms (+0 water, +1 water, +2 water) to quantify conjugate concentration

Use non-hydrolyzed form (+0 water) only to quantify conjugate concentration Conjugate Nominal Concentration (nM) 10 99 661 5285

N

Mean (nM)

Accuracy (%)

%CV

Mean (nM)

Accuracy (%)

%CV

3 3 3 3

9.86×100 9.28×101 6.33×102 5.52×103

98.6 93.7 95.8 104.4

7.2 6.5 1.9 3.3

1.04×101 9.14×101 6.28×102 5.54×103

104.0 92.3 95.0 104.8

7.8 8.2 3.8 1.0

Table 2. Plasma conjugate concentrations in mice dosed with EEB9.

12 12 28

Use sum of non-hydrolyzed and hydrolyzed forms (+0 water, +1 water, +2 water) to quantify conjugate concentration Concentration %CV (nM) 4.78×103 11 3 2.10×10 6.7 6.42×102 31


24

Use non-hydrolyzed form (+0 water) only to quantify conjugate concentration

EEB9

Time (h)

N

6 24 48

3 3 3

Concentration (nM) 4.99×103 2.01×103 5.75×102

%CV

Page 25 of 25

TOC for Analytical Chemistry O S

H N

N

Protein

+H2O

Protein

S

O

H N O

O

O

N H

O

O

H N O

O

N

N H

O +H2O

O

S

N O

O O N

Cl OCH3

CO2H H N

HO2C H S N

O

O

succinimide hydrolysis

Concentration (ug/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O O

N H OH OCH 3

EEB9

succinimide hydrolysis

Plasma Stability

120

Hydrolyzed and Non-hydrolyzed Non-hydrolyzed only

100 80 60 40 20 0 0

50 100 150 Incubation Time (h)

200


25

MS Bioanalysis of ProteinâDrug ConjugatesâThe Importance

Recommend Documents

MS Bioanalysis of ProteinâDrug ConjugatesâThe Importance