Bo An

Surfactant-Aided Precipitation/on-Pellet-Digestion (SOD) Procedure Provides Robust and Rapid Sample Preparation for Reproducible, Accurate and Sensitive LC/MS Quantification of Therapeutic Protein in Plasma and Tissues

For targeted protein quantification by liquid chromatography mass spectrometry (LC/MS), an optimal approach for efficient, robust and hi-throughput sample preparation is critical, but often remains elusive. Here we describe a straightforward surfactant-aided-precipitation/on-pellet-digestion (SOD) strategy that provides effective sample cleanup and enables high and constant peptide yields in various matrices, allowing reproducible, accurate and sensitive protein quantification. This strategy was developed using quantification of monocolnocal antibody in tissues and plasma as the model system. Surfactant treatment before precipitation substantially increased peptide recovery and reproducibility from plasma/tissue, likely because surfactant permits extensive denaturation/reduction/alkylation of proteins and inactivation of endogenous protease inhibitors, and facilitates removal of matrix components. The subsequent precipitation procedure effectively eliminates the surfactant and nonprotein matrix components, and the thorough denaturation by both surfactant and precipitation enabled very rapid on-pellet-digestion (45 min at 37 °C) with high peptide recovery. The performance of SOD was systematically compared against in-solution-digestion, in-gel-digestion and filter-aided-sample-preparation (FASP) in plasma/tissues, and then examined in a full pharmacokinetic study in rats. SOD achieved the best peptide recovery (∼21.0-700% higher than the other three methods across various matrices), reproducibility (3.75-10.9%) and sensitivity (28-30 ng/g across plasma and tissue matrices), and its performance was independent of matrix types. Finally, in validation and pharmacokinetic studies in rats, SOD outperformed other methods and provided highly accurate and precise quantification in all plasma samples without using stable isotope labeled (SIL)-protein internal standard (I.S.). In summary, the SOD method has proven to be highly robust, efficient and rapid, making it readily adaptable to large-scale clinical and pharmaceutical quantification of biomarkers or biotherapeutics.

Toward Sensitive and Accurate Analysis of Antibody Biotherapeutics by Liquid Chromatography Coupled with Mass Spectrometry

Remarkable methodological advances in the past decade have expanded the application of liquid chromatography coupled with mass spectrometry (LC/MS) analysis of biotherapeutics. Currently, LC/MS represents a promising alternative or supplement to the traditional ligand binding assay (LBA) in the pharmacokinetic, pharmacodynamic, and toxicokinetic studies of protein drugs, owing to the rapid and cost-effective method development, high specificity and reproducibility, low sample consumption, the capacity of analyzing multiple targets in one analysis, and the fact that a validated method can be readily adapted across various matrices and species. While promising, technical challenges associated with sensitivity, sample preparation, method development, and quantitative accuracy need to be addressed to enable full utilization of LC/MS. This article introduces the rationale and technical challenges of LC/MS techniques in biotherapeutics analysis and summarizes recently developed strategies to alleviate these challenges. Applications of LC/MS techniques on quantification and characterization of antibody biotherapeutics are also discussed. We speculate that despite the highly attractive features of LC/MS, it will not fully replace traditional assays such as LBA in the foreseeable future; instead, the forthcoming trend is likely the conjunction of biochemical techniques with versatile LC/MS approaches to achieve accurate, sensitive, and unbiased characterization of biotherapeutics in highly complex pharmaceutical/biologic matrices. Such combinations will constitute powerful tools to tackle the challenges posed by the rapidly growing needs for biotherapeutics development.

Effects of Calibration Approaches on the Accuracy for LC–MS Targeted Quantification of Therapeutic Protein

LC–MS provides a promising alternative to ligand-binding assays for quantification of therapeutic proteins and biomarkers. As LC–MS methodology is based on the analysis of proteolytic peptides, calibration approaches utilizing various calibrators and internal standards (I.S.) have been developed. A comprehensive assessment of the accuracy and reliability of these approaches is essential but has yet been reported. Here we performed a well-controlled and systematic comparative study using quantification of monoclonal-antibody in plasma as the model system. Method development utilized a high-throughput orthogonal-array-optimization, and two sensitive and stable signature-peptides (SP) from different domains were selected based on extensive evaluations in plasma matrix. With the purities of all protein/peptide standards corrected by quantitative amino acid analysis (AAA), five calibration approaches using stable-isotope-labeled (SIL) I.S. were thoroughly compared, including those at peptide, extended-peptide, and protein levels and two “hybrid” approaches (i.e., protein calibrator with SIL-peptide or SIL-extended-peptide I.S.). These approaches were further evaluated in parallel for a 15 time point, preclinical pharmacokinetic study. All methods showed good precision (CV% < 20%). When examined with protein-spiked plasma QC, peptide-level calibration exhibited severe negative biases (−23 to −62%), highly discordant results between the two SP (deviations of 38–56%), and misleading pharmacokinetics assessments. Extended-peptide calibration showed significant improvements but still with unacceptable accuracy. Conversely, protein-level and the two hybrid calibrations achieved good quantitative accuracy (error < 10%), concordant results by two SP (deviations < 15%), and correct pharmacokinetic parameters. Hybrid approaches were found to provide a cost-effective means for accurate quantification without the costly SIL-protein. Other key findings include (i) using two SP provides a versatile gauge for method reliability; (ii) evaluation of peptide stability in the matrix before SP selection is critical; and (iii) using AAA to verify purities of protein/peptide calibrators ensures accurate quantitation. These results address fundamental calibration issues that have not been adequately investigated in published studies and will provide valuable guidelines for the “fit for purpose” development of accurate LC–MS assays for therapeutic proteins and biomarkers in biological matrices.

Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry

In the last decade, the advancement of liquid chromatography mass spectrometry (LC/MS) techniques has enabled their broad application in protein characterization, both quantitatively and qualitatively. Owing to certain important merits of LC/MS techniques (e.g., high selectivity, flexibility, and rapid method development), LC/MS assays are often deemed as preferable alternatives to conventional methods (e.g., ligand-binding assays) for the analysis of protein biotherapeutics. At the discovery and development stages, LC/MS is generally employed for two purposes absolute quantification of protein biotherapeutics in biological samples and qualitative characterization of proteins. For absolute quantification of a target protein in bio-matrices, recent work has led to improvements in the efficiency of LC/MS method development, sample treatment, enrichment and digestion, and high-performance low-flow-LC separation. These advances have enhanced analytical sensitivity, specificity, and robustness. As to qualitative analysis, a range of techniques have been developed to characterize intramolecular disulfide bonds, glycosylation, charge variants, primary sequence heterogeneity, and the drug-to-antibody ratio of antibody drug conjugate (ADC), which has enabled a refined ability to assess product quality. In this review, we will focus on the discussion of technical challenges and strategies of LC/MS-based quantification and characterization of biotherapeutics, with the emphasis on the analysis of antibody-based biotherapeutics such as monoclonal antibodies (mAbs) and ADCs.

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

Quantitative proteomic profiling of paired cancerous and normal colon epithelial cells isolated freshly from colorectal cancer patients

The heterogeneous structure in tumor tissues from colorectal cancer (CRC) patients excludes an informative comparison between tumors and adjacent normal tissues. Here, we develop and apply a strategy to compare paired cancerous (CEC) versus normal (NEC) epithelial cells enriched from patients and discover potential biomarkers and therapeutic targets for CRC.

Catch-and-Release anti-CEA mAb leads to greater plasma and tumor exposure in a mouse model of colorectal cancer

In this study, we examined the effects of target expression, neonatal Fc receptor (FcRn) expression in tumors, and pH-dependent target binding on the disposition of monoclonal antibodies (mAbs) in murine models of colorectal cancer. A panel of anti-carcinoembryonic antigen (CEA) mAbs was developed via standard hybridoma technology and then evaluated for pH-dependent CEA binding. Binding was assessed via immunoassay and radioligand binding assays. 10H6, a murine IgG1 mAb with high affinity for CEA at pH = 7.4 (KD = 12.6 ± 1.7 nM) and reduced affinity at pH = 6.0 (KD = 144.6 ± 21.8 nM), and T84.66, which exhibits pH-independent CEA binding (KD = 1.1 ± 0.11 and 1.4 ± 0.16 nM at pH 7.4 and 6.0), were selected for pharmacokinetic investigations. We evaluated pharmacokinetics after intravenous administration to control mice and to mice bearing tumors with (MC38CEA+, LS174T) and without (MC38CEA−) CEA expression and with or without expression of murine FcRn, at doses of 0.1, 1, and 10 mg/kg. 10H6 displayed linear pharmacokinetics in mice bearing MC38CEA+ or MC38CEA− tumors. T84.66 displayed linear pharmacokinetics in mice with MC38CEA− tumors but dose-dependent nonlinear pharmacokinetics in mice bearing MC38CEA+. In addition to the improved plasma pharmacokinetic profile (i.e., linear pharmacokinetics, longer terminal half-life), 10H6 exhibited improved exposure in MC38CEA+ tumors relative to T84.66. In mice bearing tumors with CEA expression, but lacking expression of murine FcRn (LS174T), 10H6 demonstrated nonlinear pharmacokinetics, with rapid clearance at low dose. These data are consistent with the hypothesis that pH-dependent CEA binding allows mAb dissociation from target in acidified endosomes, enabling FcRn-mediated protection from target-mediated elimination in mice bearing MC38CEA+ tumors.

Surfactant Cocktail-Aided Extraction/Precipitation/On-Pellet Digestion Strategy Enables Efficient and Reproducible Sample Preparation for Large-Scale Quantitative Proteomics

For quantitative proteomics, efficient, robust, and reproducible sample preparation with high throughput is critical yet challenging, especially when large cohorts are involved, as is often required by clinical/pharmaceutical studies. We describe a rapid and straightforward surfactant cocktail-aided extraction/precipitation/on-pellet digestion (SEPOD) strategy to address this need. Prior to organic solvent precipitation and on-pellet digestion, SEPOD treats samples with a surfactant cocktail (SC) containing multiple nonionic/anionic surfactants, which achieves (i) exhaustive/reproducible protein extraction, including membrane-bound proteins; (ii) effective removal of detrimental nonprotein matrix components (e.g., >94% of phospholipids); (iii) rapid/efficient proteolytic digestion owing to dual (surfactants + precipitation) denaturation. The optimal SC composition and concentrations were determined by Orthogonal-Array-Design investigation of their collective/individuals effects on protein extraction/denaturation. Key parameters for cleanup and digestion were experimentally identified as well. The optimized SEPOD procedures allowed a rapid 6 h digestion providing a clean digest with high peptide yields and excellent quantitative reproducibility (especially low-abundance proteins). Compared with filter-assisted sample preparation (FASP) and in-solution digestion, SEPOD showed superior performance by recovering substantially more peptide/proteins (including integral membrane proteins), yielding significantly higher peptide intensities and improving quantification for peptides with extreme physicochemical properties. SEPOD was further applied in a large-cohort temporal investigation of 44 IAV-infected mouse lungs, providing efficient and reproducible peptide yields (77.9 ± 4.6%) across all samples. With the IonStar pipeline, >6 400 unique protein groups were quantified (≥2 peptide/protein, peptide-FDR < 0.05%), ∼99% without missing data in any sample with <7% technical median-intragroup CV. Altered proteome patterns revealed interesting novel insights into pathophysiological changes by IAV infection. In summary, SEPOD offers a feasible solution for rapid, efficient, and reproducible preparation of biological samples, facilitating high-quality proteomic quantification of large sample cohorts.