A Sensitive and Reproducible Signature Peptide MRM Based Quantitation Method for Pegylated Interferon α-2b in Serum

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1 A Sensitive and Reproducible Signature Peptide MRM Based Quantitation Method for Pegylated Interferon α-2b in Serum SCIEX QTRAP 6500 LC-MS/MS System Faraz Rashid 1,Dipankar Malakar 1, Anoop Kumar 1,Manoj Pillai 1,Praveen Kumar V 2, Manoj Bob 2, Ravisekhar K 2 1 SCIEX, 121, Udyog Vihar, Phase IV, Gurgaon, Haryana, India 2 Lupin bioresearch Center, Pune Maharasthra, India Key Challenges of Pegylated Interferon α-2b Quantitation Sensitivity: The overlapping protein charge state distribution and the poly-dispersity of the pegylation complicate the ESI-MS spectrum and dilute signal intensity making intact quantitation at sub pg levels difficult to achieve Sample Clean-up: Selective isolation of the low concentration protein drug from the abundant plasma endogenous proteins without loss is difficult Interferences: Immunological approaches to this assay are available however, imprecision and matrix interferences are common issues Unique Features of this Procedure for Quantification of Pegylated Interferon α-2b from Serum High Sensitivity: An LLOQ of 246 pg/ml in serum is achieved using signature peptide detection and selective sample cleanup. Maximum sensitivity is achieved by the patented IonDrive TM technology of the SCIEX QTRAP 6500 System. Linear Dynamic Range (LDR): A dynamic range from 246 to 50,000 pg/ml from serum samples is supported by the IonDrive High Energy Detector technology. Easy Sample Processing: Removal of high abundance endogenous proteins is achieved by selective protein precipitation followed by enzymatic digestion. INTRODUCTION Figure 1. The SCIEX QTRAP 6500 System. lysine residues. Pegylation is one of the most effective and acceptable procedures for stabilization of therapeutic proteins. This stabilization procedure is important to sustain absorption, generate a longer half-life and shorten renal clearance 1,2,3. In this technical note, we report an LC-MS/MS signature peptide based method for the quantification of PEG-IFN- α-2b in serum that can be used in bioanalytical labs. As reported earlier by Yang Z, et al, LOQ of 3.6ng/ml was achieved for pegylated-interferon-α-2a. To achieve maximum sensitivity several parameters related to sample processing, chromatography and mass spectrometric parameters were optimized. Extraction of PEG-IFN- α-2b was obtained through precipitation of endogenous proteins in serum followed by enzymatic digestion of supernatant. A surrogate tryptic peptide generated from the pegylated protein was used for MRM based quantitation at picogram per ml levels in serum using the SCIEX QTRAP 6500 system. Pegylated drug conjugate Interferon α-2b (PEG-IFN- α-2b) is a 165 amino acid therapeutic protein used for the treatment of chronic hepatitis C infection. Pegylated interferon α-2b is produced by covalent attachment of 40 kda PEG (polyethylene-glycol) to interferon-α2b via the p 1

2 Materials and Methods PEG-IFN- α-2b was obtained commercially (lyophilized powder) and diluted in aqueous buffer containing 150mM ammonium formate ph 4.5. Table 1. MS Parameters for MRM Quantitation Name Q1 Q3 Dwell DP CE CXP Peptide Peptide Peptide Table 2. Gradient profile for MRM Quantitation Column Mobile Phase A Mobile Phase B Flow rate Atlantis dc18, 100x 2.1 mm, 3.5μm Water, 0.1% formic acid Methanol, 0.1% formic acid 400 µl/min Column temperature 45 C Injection volume 30 µl Gradient profile Time (min) % B Gradient Conditions for Signature Peptide Selection A Shimadzu Nexera LC system was used with an Atlantis-dC18, 100x 2.1 mm, 3.5μm column to separate the tryptic digest at a flow rate of 300 µl/min. The tryptic peptides were chromatographically separated with a gradient starting at 5% acetonitrile (0.1% formic acid) to 40% of acetonitrile (0.1% formic acid) in 35 min run at 40 C. Mass Spectrometry Conditions The SCIEX QTRAP 6500 system with Turbo V Ion Drive source and electrospray ionization (ESI) probe was used for peptide selection and quantitation experiments. The QTRAP based Information Dependent Acquisition (IDA) workflow was used to identify a unique peptide from PEG-IFN- α-2b and sequence confirmation was done by Protein Pilot TM 5.0. Additionally, Skyline software was used to identify the right precursor and its fragments followed by method creation. Peptide sequence SFSLSTNLQESLR (m/z >1047.5) was identified as most stable and ionized sequence ion from the PEG- IFN- α-2b. Three transitions ( >1047.5, and 745.4) were selected for data acquisition but only ( >1047.5) transition was used for quantitation. To achieve maximum sensitivity both the source and compound related parameters were optimized Table 3. Sample Preparation Step 1 Isopropyl alcohol (600 ul) contaiuning 0.1% formic acid was added to 200 ul of plasma and gently mixed for 10 minutes at room temperature. Step 2 Samples were centrifuged at 4000 rpm for 10 min at 5 C to collect the precipitate and the supernatant was then removed to a fresh set of tubes and dried under N2. Step 3 The dried supernatant was reconstituted in 300 ul of digest buffer of 10 mm ammonium bicarbonate, 50% methanol and 5 ul BSA Step 4 Trypsin 10 ul, 1 mg/ml was addded and the digestion was left overnight at 37 C Step 5 The digestion reaction was quenched with 10 ul of 10% formic acid. Step 6 After centrifugation to collect any precipitate the samples were injected. Results and Discussion Improving sensitivity is one of the major challenges for the application of MS-based assays for protein bioanalysis. The existence of highly abundant endogenous proteins in biological matrices may cause high background noise and ion suppression during MS detection, which will significantly reduce the sensitivity of the assay. Methods for sample preparation that can remove the high abundance background proteins and selectively extract the target protein are critical to overcome these challenges. To improve the assay sensitivity and selectivity a variety of sample preparation strategies have been applied to the purification of the target protein prior to digestion. Protein precipitation can be used for the extraction of organic soluble proteins (e.g., pegylated proteins), since they are retained in the supernatant and can be separated from other precipitated proteins (Wu et. al., 2011). The advantage of this approach is that protein precipitation can provide some degree of sample cleanup by removing soluble proteins, salts, and a significant portion of phospholipids. We have optimized the method with IPA with formic acid assisted protein precipitation that can efficiently remove albumin, the most abundant endogenous protein in serum samples, while retain the p 2

3 target protein, thereby obtaining cleaner samples and achieving improved sensitivity. ELISAs are the most popular analytical method for protein quantification, but can be challenging to use for PEGylated because epitope-masking may limit sensitivity and accuracy. LC-MS/MS based quantitation is becoming more popular because of its advantage of selecting specific signature peptides in complex drug conjugate protein molecules. A sensitive and reproducible LC- MS/MS method for PEG-IFN- α-2b has been developed in this study. The sample extraction and cleanup workflow is described in Table 3. The total ion chromatogram (TIC) of tryptic digested PEG-IFN- α-2b and extracted ion chromatogram (XIC) of the selected signature peptide SFSLSTNLQESLR using a non-targeted IDA workflow with QTRAP technology is depicted in Figure 2. The calibration curve in serum extracted samples showed excellent linearity with an r value of >0.99 for the linear range between ,000 pg/ml. Chromatograms of various calibration samples are given in Figure 3. The chromatograms of extracted serum blank, HQC and low limit of quantitation (LLOQ) are shown in Figure 4. The LLOQ has an excellent signal to noise ratio of Three precision and accuracy batches were processed and tested. The QC sample statistics are summarized in Table 4. All the three batches were within the recommended acceptance criteria for protein LC-MS/MS of %CV ± 25% of LLOQ level and ±20% at other levels. 6 The recovery of the PEG-IFN- α-2b signature peptide was 76% across all QC concentrations. Table 4. The accuracy of PEG-IFN- α-2b at different concentration levels in serum. % Accuracy PEG-IFN- α-2b Statistics LLQC LQC MQC HQC N Mean S.D % C.V % Accuracy serum matrix. This sample preparation approach is less time consuming and more affordable in CRO business. Linearity within a concentration range of ,000 pg/ml of PEG-IFN- α-2b was achieved in serum matrix with regression coefficients r References 1. Rinat R. et al., Structural characterization of protein polymer conjugates. I. Assessing heterogeneity of a small PEGylated protein and mapping conjugation sites using ion exchange chromatography and topdown tandem mass spectrometry. International Journal of Mass Spectrometry 312 (2012) Wu S, et al, A strategy for liquid chromatography /tandem mass spectrometry based quantitation of pegylated protein drugs in serum using serum protein precipitation with water-miscible organic solvents and subsequent trypsin digestion to generate surrogate peptides for detection. Rapid Commun Mass Spectrom. 2011; 25(2): Yang Z, et al., A sensitive and high-throughput LC- MS/MS method for the quantification of pegylatedinterferon-alpha2a in human serum using monolithic C18 solid phase extraction for enrichment. J Chromatogr B. 2009; 877(18-19): Long Yuan and Mingshe Zhu, Quantitative Bioanalysis of Proteins by Mass Spectrometry. Materials and Methods 2015;5: Bioanalysis-of-Proteins-by-Mass-Spectrometry.html 5. Catherine François, et al., Quantification of Different Human Alpha Interferon Subtypes and Pegylated Interferon Activities by Measuring MxA Promoter Activation. Antimicrob Agents Chemother Sep; 49(9): Rand Jenkins, et al., Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics. AAPS J Jan; 17(1): Conclusions A highly specific and robust LC-MS/MS based quantitation method has been developed for the pegylated drug conjugate Interferon-α2b in serum. A doubly charged signature peptide was selected for better and robust ionization and used for MRM quantitation m/z => A liquid-liquid extraction (LLE) based method was optimized for the quantitation of PEG-IFN- α-2b from p 3

4 Figure 2: Total ion chromatogram (TIC) of tryptic digested Pegylated Drug Conjugate Interferon-α2b (a) and extracted ion chromatogram (XIC) of selected peptide SFSLSTNLQESLR (b) using non-targeted IDA workflow on QTRAP technology. Figure 3: Chromatograms of extracted peptide SFSLSTNLQESLR in serum. p 4

5 BLANK HQC (60,000 pg/ml) LLOQ (246.0 pg/ml) Figure 4: Chromatograms of blank serum, LLOQ and HQC samples. AB Sciex is doing business as SCIEX AB Sciex. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Document number: RUO-MKT B p 5