High-Throughput Method Development for Sensitive, Accurate, and Reproducible Quantification of Therapeutic Monoclonal Antibodies in Tissues Using Orthogonal Array Optimization and Nano Liquid Chromatography/Selected Reaction Monitoring Mass Spectrometry

Abstract

Although liquid chromatography/mass spectrometry using selected reaction monitoring (LC/SRM-MS) holds great promise for targeted protein analysis, quantification of therapeutic monoclonal antibody (mAb) in tissues represents a daunting challenge due to the extremely-low tissue levels, complexity of tissue matrices, and the absence of an efficient strategy to develop an optimal LC/SRM-MS method. Here we describe a high-throughput, streamlined strategy for the development of sensitive, selective and reliable quantitative methods of mAb in tissue matrices. A sensitive nano-LC/nanospray-MS method was employed to achieve a low lower limit of quantification (LOQ). For selection of signature peptides (SP), the SP candidates were identified by a high-resolution Orbitrap and then optimal SRM conditions for each candidate were obtained using a high-throughput, on-the-fly orthogonal array optimization (OAO) strategy, which is capable of optimizing a large set of SP candidates within a single nano-LC/SRM-MS run. Using the optimized conditions, the candidates were experimentally evaluated for both sensitivity and stability in the target matrices and SP selection was based on the results of the evaluation. Two unique SPs, respectively from the light and heavy chain, were chosen for quantification of each mAb. The use of two SP improves the quantitative reliability by gauging possible degradation/modification of the mAb. Standard mAb proteins with verified purities were utilized for calibration curves, to prevent the quantitative biases that may otherwise occur when synthesized peptides were used as calibrators. We showed a proof of concept by rapidly developing sensitive nano-LC/SRM-MS methods for quantifying two mAb (8c2 and cT84.66) in multiple preclinical tissues. High sensitivity was achieved for both mAb with LOQ ranged from 0.156 to 0.312 μg/g across different tissues, and the overall procedure showed a wide dynamic range (≥500 fold), good accuracy (RE<18.8%) and precision (inter-batch RSD<18.1%, intra-batch RSD<17.2%). The quantitative method was applied to a comprehensive investigation of the steady-state tissue distribution of 8c2 in wild-type mice vs. those deficient in FcRn α-chain, FcγIIb, and FcγRI/FcγRIII, following a chronic dosing regimen. This work represents the first extensive quantification of mAb in tissues by an LC/MS-based method.

Proteomic Analysis Reveals Diverse Classes of Arginine Methylproteins in Mitochondria of Trypanosomes

“…2A and 2C. The symmetry-specific neutral losses from ADMA and SDMA have been well-characterized by our lab and others (25,27,28). Moreover, CID was helpful for some peptides that have lower charge states (supplemental Table S1).…”

“…A strong, detergent-containing buffer was used to obtain excellent recovery of proteins, especially those that are membrane-bound, as demonstrated in our previous works (22,25). This was followed by efficient precipitation/on-pelletdigestion using two enzymes with orthogonal specificity (22), trypsin (which cuts at the C terminus of lysine and arginine) and GluC (which cuts at the C terminus of glutamic acid and aspartic acid), to digest the mitochondrial preparations in parallel.…”

Ion-current-based Proteomic Profiling of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome

“…An Orbitrap analyzer with an "overfilling" approach to enhance sensitivity without compromising MS accuracy and resolution (26,35,36) was used to produce the precursor ion currents for quantification. Using a low-void-volume and reproducible nano-LC/nanospray configuration modified from what we had developed previously (22,26,35,37), we extensively resolved retinal samples on a long, heated reversephase nano-LC column (75 cm long with 3-m particles and heated at 52°C) with a shallow, 7-h gradient. A wide peptide elution window (Ͼ315 min) with an average peak width of less than 30 s (full width at half-maximum) and a peak capacity greater than 600 was achieved, which enabled in-depth profiling of the retinal proteome.…”

“…To address this problem, we homogenized retinas using a Polytron tissue homogenizer in a "strong" buffer containing a mixture of detergents (2% Nonidet P-40, 2% SDS, and 0.5% sodium deoxycholate) that has been previously demonstrated by our lab to efficiently extract proteins from biological tissues, including membrane proteins (22,26,35,36). The extracted proteins were subjected to a quantitative precipitation/on-pellet-digestion procedure (22,26,37) to remove detergents and other non-protein components and then completely digest the extracts. Under the optimized conditions, high, reproducible protein yields were achieved (supplemental Fig.…”

Current Mass Spectrometric Tools for the Bioanalyses of Therapeutic Monoclonal Antibodies and Antibody-Drug Conjugates

“…39 Thus, searching the signature peptide and optimizing the MS/MS condition demands much labor and time. To speed up these tasks, Duan and colleagues have reported a new methodology, 41 in which the tissue samples, spiked with target mAbs are digested with trypsin, and a fragment thereof is analyzed by a high resolution mass spectrometer (HRMS) Orbitrap in the presence of the sample matrix. Then, by combining the nano LC/MS/MS and the direct sequence method, the SRM parameters for the identified signature peptide are optimized by an analysis.…”