Generic Automated Method for Liquid ChromatographyâMultiple

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A Generic Automated Method for LC-MRM-MS Based Monoclonal Antibody Quantitation for Pre-Clinical Pharmacokinetic Studies Qian Zhang, Daniel S Spellman, Yaoli Song, Bernard K. Choi, Nathan G. Hatcher, Daniela Tomazela, Maribel Beaumont, Mohammad Tabrizifard, Deepa Prabhavalkar, Wolfgang Seghezzi, Jane Harrelson, and Kevin P. Bateman Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5019827 • Publication Date (Web): 10 Jul 2014 Downloaded from http://pubs.acs.org on July 14, 2014

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Analytical Chemistry

A Generic Automated Method for LC-MRM-MS Based Monoclonal Antibody Quantitation for Pre-Clinical Pharmacokinetic Studies

Qian Zhang1, Daniel S. Spellman1, Yaoli Song2, Bernard Choi3, Nathan G. Hatcher1, Daniela Tomazela2, Maribel Beaumont2, Mohammad Tabrizifard2, Deepa Prabhavalkar2, Wolfgang Seghezzi2, Jane Harrelson1, Kevin P. Bateman1,* 1. Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Inc., West Point, PA 2. Biologics Discovery, Merck & Co., Inc. Palo Alto, CA 3. Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Inc., Rahway, NJ Correspondence: [email protected]

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Abstract Quantitation of therapeutic monoclonal antibodies (mAb) using LC-MS/MS for pharmacokinetic (PK) studies is becoming an essential complement to traditional antibody-based ligand binding assays (LBA). Here we show an automated method to perform LC-MS/MS based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard, and anti-human Fc enrichment. All reagents in the method are commercially available with no requirement to develop novel assay-specific reagents. The method met traditional quantitative LC-MS/MS assay analytical characteristics in terms of precision, accuracy and specificity. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey and the results were compared with the immunoassay data. This methodology has the potential to benefit and accelerate the early biopharmaceutical development process, particularly by enabling PK analysis across species and candidate molecules with minimal method development.

Introduction Beginning with the FDA approval of recombinant insulin in 1982, biotherapuetics, products of which the active substance is produced by or extracted from a biological source, have represented an important and growing constituent of prescribed drugs. Therapeutic monoclonal antibodies (mAbs) represent the majority of biotherapeutics now on the market, with more than 20 mAbs approved as drugs by the FDA over the past 30 plus years 1-7. The majority of the marketed mAb’s are based on the IgG isotype subclass 1 (IgG1). During preclinical development of therapeutic antibodies, multiple variants of each antibody are assessed for pharmacokinetic (PK) characteristics across model systems such as rodents and non-human primates. During clinical development, PK studies are also required for the mAb drug in human matrices. Traditionally, ligand-binding assays (LBA) are used to perform the PK studies for mAb candidates 8. However, LBAs have certain well documented limitations9. Specific assay reagents are often not available early on in a program. Interferences from endogenous proteins, antidrug antibodies, and soluble target ligands are potential complicating factors. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) based methods represent a viable and complementary addition to LBA for mAb quantification in biological matrixes. LC-MS/MS provides specificity, sensitivity, and multiplexing capability in a largely matrix-independent fashion. Specifically, LC coupled with triple quadruple (QQQ) based multiple reaction monitoring (MRM) 10 is the gold standard for quantitative analysis in the pharmaceutical industry. Recently, several LC-MS/MS-based methods have been developed for the quantitation of mAbs representing a significant advance in PK sample analysis on therapeutic mAbs 9, 11-17. Most of these methods employ enzymatic digestion of the mAb and one or more surrogate peptides with unique sequences are chosen for quantification, representing the whole mAb. Multidimensional chromatography or immuno-enrichment can be used to separate the mAb of interest from those of the


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endogenous matrix, which results in increased sensitivity due to lower background and concentration of the analyte of interest 11, 12, 18, 19. Here we show a generic LC-MRM-MS based method, employing broadly conserved peptides, a heavy isotope labeled representative mAb internal standard, and anti-human Fc enrichment, using readily available commercial reagents. The sample preparation method was implemented on a Hamilton Starlet platform to automate the immuno-affinity purification of the target mAb. The method is applied to the quantification of a candidate therapeutic mAb dosed in a non-human primate PK study and results are compared to LBA data. The generic method can be applied for the quantification of IgG1 based therapeutic antibodies across pre-clinical studies.



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Experimental Chemicals and Reagents. The Merck molecules αIL-17, αIL-10 and αPCSK9 were prepared at Merck Research Laboratories. Commercial Remicade was obtained from Janssen Biotech. The Stable-isotope labeled universal monoclonal antibody (SILUMAb) standard was obtained from Sigma, incorporating [13C, 15N]lysine/arginine. Animal plasma and serum (rat, mouse, guinea pig, rabbit, beagle, cynomolgus monkey) were obtained from Bioreclamation Inc. (East Meadow, NY). The goat anti-human IgG antibody (monkey adsorbed) was purchased from SouthernBiotech (Birmingham, AL). The streptavidin magnetic beads were purchased from Invitrogen (Carlsbad, CA). The Tris-buffered saline (TBS) and Tris-Cl buffers were purchased from bioWORLD (Dublin, OH). DTT was purchased from Pierce (Rockford, IL). Iodoacetamide, guanidine, urea, formic acid, trifluoroacetic acid, trypsin from bovine pancreas (TPCK treated), water (HPLC grade) and methanol (MeOH) were purchased from Sigma (St. Louis, MO). Sequencing grade trypsin (TPCK treated) was purchased from Promega (Madison, WI). HPLC solvent acetonitrile and water with 0.1% FA were purchased from Fisher Scientific (Waltham, MA). Screening for Surrogate Peptides. The mAbs (αIL-17, αIL-10 and Remicade) were diluted to 0.33mg/mL in a total volume of 400 µL of 6.0 M Guanidine-HCl in 0.1M Tris-HCl, pH7.6 in an Eppendorf tube. DTT was added to reach 5 mM final concentration. The samples were incubated for 30 minutes at 37 ºC followed by cooling down to room temperature. Iodoacetamide was added to reach 12 mM final concentration and the samples were incubated in the dark for 30 minutes at room temperature. After applying an AmiconUltra 0.5 mL 30K filter, the samples were cleaned and concentrated to 20 µL, followed by reconstitution in 400 µL of 250 mM Tris-HCl, pH 7.5. The samples were then mixed with 10 µL of 1 µg/µL sequencing grade trypsin and digested at 37 °C overnight. Digested samples were analyzed by a reverse phase nano-UPLC (Waters nanoAcquity) coupled to a Velos Orbitrap hybrid mass spectrometer with electron transfer dissociation (ThermoFisher). Two microliters of digested sample was loaded onto a capillary sample trap column (100 um ID, 2.5 cm; ProteoPrep 2; IntegraFrit; New Objectives) and desalted on line for 5 min at 3 µL/min with 99.5% solvent A [100 % HPLC grade water, 0.1 % formic acid] 0.5% solvent B [100 % HPLC grade acetonitrile, 0.1 % formic acid]. After 5 minutes the flow rate was reduced to 0.25 µl/min and peptides were eluted into packed spray tip column (75 µm i.d., 190µm o.d. × 10 cm; Reprosil-Pur C18; PicoFrit 15 µm; New Objectives). Bound peptides were eluted from the column via a linear gradient from 0.1 to 35% solvent B over 80 minutes followed by an isocratic gradient of 5 minutes at 90% solvent B and 5 minutes at 0.1% solvent B. Eluted peptides were introduced to the Velos Orbitrap via nanoelectrospray and high resolution mass spectra were acquired at a rate of 1 spectrum per second. These spectra recorded the intensity, retention time, and accurate mass-to-charge ratio for the discrete peptide ions in each sample. 15 data-dependent MS/MS spectra were acquired for the most intense ions in each full scan spectra to provide amino acid sequence information for selected peptide ions. The data was analyzed by X! Tandem database search (details in supplemental method section) to identify ideal surrogate peptides.


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LC-MRM-MS Instrumentation The sequences of selected surrogate peptides were imported to Skyline 20 to generate MRM instrument methods. The transitions were selected based on the prediction of Skyline and the experimental results of the high resolution data dependent LC-MS/MS data. Skyline used a spectral library to rank the fragment ions from target peptides by intensity and selected transitions as reported previously 21. The LC-MRM-MS experiments were performed on an automated LC-MS/MS system which consisted of an Acquity I-Class UPLC system (Waters, Milford, MA) coupled to a Xevo TQ-S tandem quadrupole mass spectrometer (Waters, Milford, MA). The analytical column was an Acquity UPLC BEH Shield RP C18 2.1 mm × 100 mm column with 1.7 μm particle size. The mobile phases were 0.1% formic acid in H2O (mobile phase A) and 0.1% formic acid in ACN (mobile phase B). The LC gradient was performed as follows (min/% of mobile phase B): 0.0/2, 0.2/2, 8.0/50, 12/90, 14/2 and 16/2. Total runtime was 18 min. The flow rate was kept at 0.30 mL/min, and the column temperature was 60 °C. Data was collected by MassLynx (version 4.1) and processed by Skyline. The ESI spray voltage was set at 3.0 kV for the z-spray source. The source temperature was 150 °C and the desolvation temperature was 300 °C. The cone gas flow was 150 L/Hr, the desolvation gas flow was 500 L/Hr and the collision gas flow was 0.24 mL/Min. The MRM specifics for each optimized surrogate peptides are listed in Table 1. Plasma Pellet Digestion The digestion protocol was modified from the procedure reported by Ouyang et al22. Briefly, 50 μL of plasma sample was mixed with 50 μL of 100 mM ammonium bicarbonate in water and then incubated at 90°C for 25 min on a shaker. Then 20 μL of 100 mM DTT in water was added; followed by incubation at 60°C for 1 h. After cooling to room temperature, 10 μL of 100 mM iodoacetamide in water was added, and the mixture was incubated at 30°C in dark for 30 min. The treated sample was mixed with 200 μL of methanol, followed by centrifugation at 900 × g for 5 min at 4 °C. The supernatant was removed carefully, and the protein pellets were mixed with 200 μL of 200 mM ammonium bicarbonate in 10% MeOH/H2O. Then 50 μL of trypsin (TPCK treated trypsin from bovine pancreas, Sigma) in 100 mM ammonium bicarbonate (8 mg/ml, freshly prepared) were added to each sample. After gently mixing, the mixture was incubated at 60°C with shaking at 750 rpm for one hour. Then the tryptic digests were mixed with 5 μL of formic acid in water and centrifuged at 5000 × g at 4°C for 10 min. The supernatant was analyzed by LC-MRM-MS. Immuno-Precipitation and Digestion Streptavidin magnetic beads were washed 3 times with buffer Tris-buffered saline (TBS). Biotinylated goat anti-human-IgG antibody was mixed with the beads for 30 minutes at room temperature on a rotating device. An 800 μL antibody solution (0.5 mg/ml) was used to label ~10 mg beads. The ~10 mg coated beads were washed 3 times in buffer TBS and reconstituted in 1 ml buffer TBS. For the standard curve, each sample was prepared by mixing 25 μL of cynomolgus monkey serum, 25 μL of aPCSK9 solution (with 10 different concentrations covering the range of 0 to 25 μg/ml), 25 μL of IS solution (3 μg/ml SILUMAb) and 175 μL buffer TBS. For the cynomolgus monkey PK samples, 25 μL of serum is mixed with 25 μL of internal standard (IS) solution and 200 μL buffer TBS.



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The prepared sample with a total volume of 250 μL was then mixed with 100 μL (1 mg) coated beads in a 96-well plate. A Hamilton Starlet liquid handler (Reno, NV) was programmed to perform the experiments with the beads. Briefly, the beads were incubated for 1 hour at room temperature with shaking to allow the affinity pull-down, washed 2 times with buffer TBS, and incubated 10 minutes with 50 μL of 0.1% TFA for elution. The eluate was transferred to a protein low-binding Eppendorf tube and neutralized by 4 μL of 1 M Tris-HCl buffer, pH 9. The sample was then mixed with 45 μL of 10 mM of DTT (in 8 M Urea, 250 mM Tris-HCl, pH 7.5). After incubation at 55 °C for 45 minutes, the mAbs were reduced and denatured. Twenty five microliters of 40 mM iodoacetamide (in 250 mM Tris-HCl, pH 7.5) were added and incubated in dark for 45 min at 55 °C. The digestion was performed by adding 300 μL of 1 µg/mL sequencing grade trypsin (in 250 mM Tris-HCl, pH 7.5) and incubation at 37 °C overnight. Five μL of acetic acid was added to quench the digestion reaction. The ~400 μL sample was cleaned up and concentrated by an Oasis HLB 96-well µElution SPE Plate (Waters). The SPE resin was firstly conditioned by 0.2 mL of methanol and equilibrated by 0.2 mL of water. The sample was drawn through and the resin was washed by 0.2 mL of 5% MeOH in water. The elution volume was 50 μL with MeOH. Ten microliters of the sample was injected for LC-MRM-MS analysis.

ELISA Method for Quantitation of anti PCSK9 mAb Concentration in cynomolgus monkey serum samples A mouse monoclonal anti idiotype capture antibody raised against the anti PCSK9 drug antibody was immobilized onto Nunc Maxisorp 96-well plates by overnight incubation at 4oC and a concentration of 1 μg/mL. Following a blocking step using 0.5 % BSA in PBS, wells were incubated with calibrator solutions (1.25 to 80 ng/mL), QC controls, and diluted cynomolgus serum samples, respectively (minimum 1:20; further dilutions series to bring measured concentrations into calibrator range). After a two hour incubation with gentle agitation at room temperature bound anti PCSK9 mAb was detected using a biotinylated second anti idiotype antibody previously shown to form an immune-sandwich in presence of anti PCSK9 mAb (data not shown), and streptavidin-HRP following standard procedures. Anti PCSK9 drug concentrations were determined using the Softmax Pro V5 software package (5 parameter logistics regression; Molecular Devices) and the plate specific standard curve.

In vivo Sample Generation PK serum samples were collected from cynomolgus monkeys after IV bolus of a 4.5 mg/mL solution (20mM Sodium acetate, 7% sucrose, pH 5.5) at doses of 3 mg/kg or 10 mg/kg aPCSK9. The samples were frozen and stored at −70 °C until analysis. Studies were conducted according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Merck & Co..


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Data Analysis for Quantitation of PK Samples The MS data was imported to Skyline software for peak area integration. Skyline calculates the area under the curve for each peak by interpolating peak height between the raw data points for the duration of the chromatographic peak elution21. The calibration curve was established from the analyte/IS peak area ratios (with peptide GPSV) with 1/concentration2 weighted linear least squares regression. The same set of PK study samples were analyzed by LC-MRM-MS and ELISA (Supplementary Methods). The aPCSK9 pharmacokinetic parameters were calculated using non-compartmental analysis (NCA) with Phoenix WinNonlin 6.3.



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Results and Discussion Surrogate Peptide Selection. After the investigation of antibody sequences from a Merck internal database, it was decided to build the initial method targeting human IgG1 antibodies. The method can be easily modified to quantify other IgGs since certain peptides are common among different IgG subclasses. The steps of the method development are shown in Figure 1. After sequence alignment and in silico digestion of the mAb sequences with Skyline, a list of 11 peptides common to human IgG1 was compiled. Peptides from the variable region were specifically avoided to enable a generic method to be developed. To screen the 11 peptides, a peptide mapping experiment for 3 different IgG1 antibodies (Merck αIL-17, αIL-10 and Remicade) was performed with the Velos Orbitrap. The MS and MS/MS spectrum for the 11 peptides were evaluated and 8 peptides with strong precursor ion and clean fragment ions were selected as potential surrogate peptides. The relative abundance of the fragment ions was also evaluated to identify 3 to 5 transitions (fragment ions monitored in a MRM experiment) for each peptide. The peptides covered both of the IgG1 heavy chain and light chain.

Experimental

Theoretical

LC-MS/MS for trypsin digested mAb

Peptides predicted by Skyline based on mAb sequences

X!tandem database search

Identify common peptides for human IgG1

Peptide mapping Peptides with high S/N

Identify peptides unique to human

Evaluate peptide’s MS1 and MS2 spectra Optimize MRM methods for selected peptide/transitions

Figure 1. The flowchart of the experimental and theoretical paths to identify the ideal surrogate peptides. The experimental high resolution MS data for the peptides enables easy selection of the


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transitions to be optimized on the triple quadruple MS 21. The theoretical screening (protein BLAST) of the peptides enables the identification of unique human peptides suitable for the generic method.

Optimization of LC-MRM-MS Conditions With the peptides and transitions selected from the high resolution data dependent LC-MS/MS data, an MRM optimization method for each peptide was exported from Skyline to the Waters Xevo TQ S instrument. The collision energy (CE) values were iterated for each transition. The values giving the best signal for the different transitions were recorded and used in the LC-MRM-MS experiments. The MRM parameters for selected peptides are as shown in Table 1. A scheduled MRM experiment tracking multiple peptides was performed for the digested mAbs (Figure 2). The relative abundance of the peptides remained constant for 3 of the tested IgG1 antibodies. The same peptides can then be applied as the surrogate peptides for any human IgG1 antibody. Among these peptides, SGTA and NQVS were reported for quantifying IgG2 mAbs23, TVAA was reported for quantifying IgG4 mAbs24 and an IgG2 homologous GPSV peptide (sequence GPSVFPLAPCSR) was also used for quantifying IgG2 mAbs23. In order to ensure accurate quantitation, two of the eight peptides were monitored but not used in the final method. The peptide SLSL is from the C-terminal of the heavy chain and is known to prone to cleavage of the lysine. Peptide GFYP is prone to deamidation.

Peptide

SLSLSPGK

NQVSLTC[+57.0]LVK

GPSVFPLAPSSK

STSGGTAALGC[+57.0]LVK

Transition 1 : 394.73 > 301.19 2 : 394.73 > 501.30 3 : 394.73 > 588.34 4 : 394.73 > 701.42 1 : 581.32 > 519.30 2 : 581.32 > 620.34 3 : 581.32 > 733.43 4 : 581.32 > 820.46 5 : 581.32 > 919.53 1 : 593.83 > 418.23 2 : 593.83 > 585.30 3 : 593.83 > 699.40 4 : 593.83 > 769.42 5 : 593.83 > 846.47 1 : 661.34 > 576.32 2 : 661.34 > 689.40 3 : 661.34 > 760.44 4 : 661.34 > 831.48

Col.Energy 19 11 11 16 23 23 23 23 23 21 21 21 21 21 25 25 24 24



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GFYPSDIAVEWESNGQPENNYK

DSTYSLSSTLTLSK

SGTASVVC[+57.0]LLNNFYPR

TVAAPSVFIFPPSDEQLK

5 1 2 3 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

: 661.34 > 932.52 : 1272.57 > 764.36 : 1272.57 > 1279.56 : 1272.57 > 1465.63 : 751.88 > 528.25 : 751.88 > 836.47 : 751.88 > 949.56 : 751.88 > 1036.59 : 751.88 > 1199.65 : 899.45 > 696.35 : 899.45 > 810.39 : 899.45 > 923.47 : 899.45 > 1036.56 : 899.45 > 1196.59 : 973.52 > 886.50 : 973.52 > 913.46 : 973.52 > 1060.53 : 973.52 > 1173.61 : 973.52 > 1320.68

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24 45 43 44 26 23 24 24 23 37 37 37 36 35 30 32 32 32 32

Table 1. The optimized MRM parameters for selected peptides. The cone voltage was 35 V for all of the peptides and the collision energy was kept the same for the transitions for labeled peptides.


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2

NQVS

1.5

GPSV 1

aIL-10

STSG

0.5

DSTY TVAA

SGTA

0 3

3.5

2

Relative Intensity (×E6)

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4

4.5

5

NQVS

1.5

GPSV 1

aIL-17

STSG

0.5

DSTY TVAA

SGTA

0 3

3.5

1.2

4

4.5

5

NQVS

1

GPSV

0.8

Remicade

0.6

STSG

0.4

DSTY

0.2

TVAA

SGTA

0 3

3.5

4

4.5

5

Time (min.)

Figure 2. The MRM experiment for 3 different mAbs. The total ion chromatogram of the monitored peptides indicates that the peptides can reproducibly represent different antibodies.

Identification of Potential Interference from Animal Serum To accommodate the need for preclinical PK studies, plasma from different species was tested for the surrogate peptides to identify potential background interferences. Briefly, the proteins from the blank (no exogenous mAb added) plasma were precipitated as a pellet, reduced, alkylated and digested by trypsin. The identified interference peaks (Table 2) are helpful in selecting surrogate peptides/transitions for each animal model system. The MRM experiments showed that the monkey plasma has significant interferences for peptide GPSV, NQVS, SLSP and STSG. In designing quantitation experiments for monkey PK studies, the peptides are carefully evaluated and only peptide GPSV was used.



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MRM transition ID

Q1 m/z

Q3 m/z

HC.GPSVFPLAPSSK.+2b6

593.83

585.30

HC.GPSVFPLAPSSK.+2b8

593.83

HC.GPSVFPLAPSSK.+2y4

Mouse

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Guinea pig

Rat

Rabbit

Beagle

Monkey

769.42

593.83

418.23


593.83

699.40


593.83

846.47

HC.NQVSLTC[+57_0]LVK.+2y4

552.81

462.27


552.81

563.32


552.81

676.41


552.81

763.44


552.81

862.51

HC.SLSLSPGK.+2y3

394.73

301.22

HC.SLSLSPGK.+2y5

394.73

501.29

HC.SLSLSPGK.+2y6

394.73

588.32

HC.SLSLSPGK.+2y7

394.73

701.39

HC.STSGGTAALGC[+57_0]LVK.+2y5

632.83

519.30


632.83

632.38


632.83

703.42


632.83

774.45


632.83

875.50

HC.GFYPSDIAVEWESNGQPENNYK.+2y12

1272.57

1465.63


1272.57

129.55


1272.57

764.36

LC.DSTYSLSSTLTLSK.+2y10

751.88

1036.59


751.88

1199.65


751.88

836.47


751.88

949.56


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LC.SGTASVVC[+57_0]LLNNFYPR.+2y5

870.94

696.35


870.94

810.39


870.94

923.47


870.94

1036.56


870.94

1139.57

LC.TVAAPSVFIFPPSDEQLK.+2b9

973.52

886.50

LC.TVAAPSVFIFPPSDEQLK.+2y10

973.52

1173.62


973.52

1320.68


973.52

913.46


973.52

1060.53

Table 2. Interference peaks identified from different animal plasma. ‘HC’ means the peptide is from the IgG heavy chain and ‘LC’ means the peptide is from the IgG light chain. ‘’ indicates an identified interference peak for the specific transition.

Consideration of Different Sample Preparation Methods The common concern for quantifying mAb using LC-MS/MS method is the sensitivity and selectivity of the method. Although a selected surrogate peptide might not have the interference peak from the animal plasma, the highly abundant endogenous proteins might still introduce high background noise or signal suppression. Recently, several groups have developed LC-MS/MS based methods for PK studies with increased specificity, reduced need for reagents and faster assay development time 22-25. Those methods generally use a sample clean-up step to reduce the amount of the endogenous proteins and to enrich the analyte protein. We initially evaluated the plasma pellet digestion protocol published by Ouyang et al 22. The lowest concentration of the antibody detected was 5 µg/ml (in 50 µl mouse plasma) using the strongest peak from peptide NQVS. To improve assay sensitivity we designed a method using a general affinity capture of protein A/G magnetic beads. The protein A/G based enrichment only improved the LLOQ to ~2 µg/ml (Data not shown). The lack of improvement of this approach relative to pellet digestion is likely due to the capacity of the beads given the high abundance of IgG’s in serum. Increasing the amount of beads to improve capture of the target mAb was not deemed viable given the cost associated with this approach. To further improve the sensitivity of the assay, a more specific anti-human Fc antibody pull down approach was evaluated 23. To eliminate the cross reactivity of the anti-human Fc antibody with the



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cynomolgus monkey IgGs, we selected a monkey absorbed commercial antibody with minimal reactivity. Unlike the result from blank plasma pellet digestion, the peptide GPSV showed no interference peak from monkey serum after the enrichment. Evaluation of Internal Standard For LC-MS/MS based quantitation, a synthetic stable isotope labeled internal standard of the surrogate peptide is often used to correct for variability in chromatography and MS ionization and enable calculation of absolute abundance. However, for large proteins (such as mAbs) in complex biological matrices (such as plasma and tissue) digestion efficiency can vary and a peptide internal standard may not always reliably correct for this. In addition, a surrogate peptide will not be able to compensate for variability when using immunoaffinity based sample preparation. To navigate these challenges several alternative approaches to internal standards have been described recently including; a stable isotope labeled version of the targeted mAb 14, or an isotopically labeled generic mAb that is applicable to various mAbs in different matrixes 23. We adapted the isotopically labeled generic mAb approach for our assay and evaluated the internal standard obtained from Sigma. The Sigma SILUMAb contains identical sequence for the constant region of human IgG1 heavy chain, with the incorporated [13C, 15N]-lysine/arginine. To avoid the potential contribution of the unlabeled analyte signal from the IS in the mass spectrometer, we evaluated the signal abundance for the labeled and unlabeled peptide from the trypsin digestion of the IS (Figure 3). Both of the high resolution data dependent LC-MS/MS and MRM data showed that portion of unlabeled molecule in the IS was minimal (< 1%).


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Figure 3. The mass spectrum (left) and MRM quantitation (right) for the selected peptide GPSVFPLAPSSK in SILUMAb. The signal of the labeled form (+8 Da) is more than 100 fold higher than the unlabeled form. The low abundance of the unlabeled peak indicates negligible interference from the internal standard.

Automation Immunoprecipitation sample preparation is a highly labor intensive and time consuming process. In order to improve throughput, enable assay consistency, and reduce laboratory scientist resource requirements for support of large scale preclinical pharmacokinetic studies, the sample preparation was automated for this method. The automation was performed on the Hamilton Starlet liquid handling robot configured with a 96 head pipette and CORE grippers. Deck configuration is shown in Figure 4. A sample plate was first transported to the magnet station. After 3 minutes, the 96 head removed the supernatant to waste and the plate was transported back to the original position. Wash solution (buffer TBS) was added to the beads and agitated with the 96 head and transported to the shaker for one



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minute of shaking. The plate was returned to the magnet and the entire process was repeated twice. After the wash, the robot added the acid (50 μL of 0.1% TFA) to the sample to release the analyte from the beads. The sample plates are removed for offline shaking at room temperature for one hour and returned back the instrument. At the magnet station, the supernatant is transferred to the collection plate for digestion. The automation system has the capacity to process two 96 well sample plates, with each plate requiring approximately 45 minutes of processing time. Capacity can be readily scaled up to enable routine sample preparation protocols for large scale pharmacokinetic studies.

Figure 4. Hamilton Starlet configuration for immunoprecipitation sample preparation. Evaluation of the Method. With the consideration of the interference peaks from Table 2, peptide GPSV was optimized for the aPCSK9 IgG1 quantitation experiments in cynomolgus monkey serum. The NQVS peptide reported previously 23 was also tested but showed strong interference in blank serum even after the anti-human Fc antibody immunocapture step. The sensitivity (LLOQ), precision (%CV) and accuracy (%bias) were


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evaluated as shown in Figure 5. The LLOQ of 100 ng/mL (Figure 5C) was achieved for peptide GPSV. No interference was observed for GPSV in blank serum (Figure 5B). The linearity of the standard curve for peptide GPSV was evaluated (Figure 5D). The peak area ratios of the analyte peptide over IS peptide were calculated with Skyline. The curves were generated by correlating the ratios with the standard concentrations using linear regression. The correlation coefficients R2 for 6 replicate curves were above 0.99. The mean regression equation was Y = 0.199(SD=0.0107) X - 0.0526(SD=0.026) for peptide GPSV from the 6 curves. The accuracy and precision of QCs are also shown in Figure 5, using the peak area ratios of analyte/IS for both of the peptides. The peptide GPSV based method is acceptable with % bias and % CV meeting the general LBA criteria of ±20% of the nominal value. We then chose peptide GPSV for the quantitation of the aPCSK9 in the cynomolgus monkey PK samples.

Figure 5. Performance characteristics of the LC-MRM-MS method. A) IS signal from single blank, B) Analyte signal from single blank, C) LLOQ of analyte, D) Standard curve from 6 independent replicates. Percent CV and Bias are shown for low (0.5 μg/mL), medium (5 μg/mL) and high (20 μg/mL) QC (n=3) samples.



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Quantitation of PK Samples Samples from cynomolgus monkeys dosed with aPCSK9 mAb were analyzed by ELISA and LC-MRM-MS methods. The concentrations from MS based quantitation were higher compared to the concentrations from the ELISA assay for both of the dosing groups (Figure 6). For the LC-MS assay the calculated area under the curve (AUCinf) was 1875 day*μg/mL and 6016 day*μg/mL for the 3 and 10 mg/kg doses, respectively. The same samples when analyzed by immunoassay yielded 691 day*μg/mL and 2265 day*μg/mL for the 3 and 10 mg/kg doses, respectively. The mAb concentration ratio (10 mg/kg over 3 mg/kg) was 3.3 for ELISA and 3.2 for LC-MS. Concentration ratios calculated from both ELISA and LC-MS confirmed the dose proportionality of mAb in cynomolgus monkey at 3 and 10 mg/kg dose levels and were found to be in good agreement. It is not unexpected to observe differences in absolute serum concentration profiles when using different assay formats; this has been shown previously for both ligand binding and mass spectrometry based assays 9, 23, 26, 27. One possible explanation is the different immunocapture reagents used in the two assays. The ELISA assay used an anti-idiotype antibody based immunosandwich to capture and detect the aPCSK9 drug. While this assay format could preferentially recognize free anti-PCSK9 mAb that does not have the PCSK9 ligand bound to the drug, we have not formally shown that it will selectively do so. In contrast, the anti-Fc immunocapture approach used with the LC-MRM-MS method most likely captures total aPCSK9 mAb with or without the ligand. Additional experiments showed that the PCSK9 ligand is present in the same sample after the anti-Fc immunocapture step (Supplemental Figure 1) indicating that at least some of the mAb pulled down by the anti-Fc antibody was binding to the PCSK9 ligand. Further analysis is needed to confirm this hypothesis and gain a better understanding of the differences in determined drug concentrations. These findings highlight the value of using both LBA and LC-MS based assays in a complementary fashion for achieving a detailed understanding the PK of a mAb therapeutic candidate in a discovery/preclinical setting. Conclusion Methodology focused on quantifying IgG1 based mAb drugs in preclinical species was developed using anti-human IgG immunocapture and a commercially available stable labeled mAb internal standard. Surrogate peptides were identified to quantify a human IgG1 mAb, aPCSK9, in cynomolgus monkey serum. The method can easily be modified to target other IgG1 drugs and other IgG subclasses. The interesting finding of the difference between ELISA and LC-MRM-MS data indicated that those 2 methods can provide complementary information (free drug vs. total drug) regarding the drug’s PK profile. This approach offers great potential value to the biopharmaceutical development process particularly during early preclinical animal studies wherein multiple structural variants of a drug candidate may need to be evaluated in various animal species to enable prioritization for further development.


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Figure 6. The comparison of the LBA data and LC-MS data for two PK studies. The red is an animal dosed with 3mg/kg aPCSK9 and the green is an animal dosed with 10 mg/kg aPCSK9.

Supporting Information Available Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.



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(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)

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+ 13

C, 15N-Labeled

Immunoprecipitation, Digestion, and LC-MS Analysis

MS Response

Preclinical mAb Pharmacokinetic Studies

Analyte

IS

1000 Serum Conc (µg/mL)

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100 10 1 0.1

3 mpk LC-MS 3 mpk ELISA 10 mpk LC-MS 10 mpk ELISA 0 7 14 21 28 35 42 49 56 63 70 77 84 Time (day)


Generic Automated Method for Liquid ChromatographyâMultiple

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