Protein drugs encompass therapeutic proteins (enzymes and regulatory proteins), targeted proteins (antibody drugs), protein vaccines, and diagnostic proteins. Among them, therapeutic proteins and antibody drugs are primarily employed in the treatment of malignant tumors, autoimmune diseases, metabolic disorders, and cardiovascular diseases.
In comparison to small-molecule drugs, protein drugs exhibit unique advantages. They possess high specificity in both structure and function, resulting in precise therapeutic effects and minimal adverse reactions. Consequently, they are highly favored. Between 2014 and 2022, protein drugs accounted for 28.5% (111 out of 389) of all drugs approved by the U.S. FDA. Monoclonal antibody drugs dominated the protein drug market, with 8 antibody drugs approved in 2022, constituting approximately 21.6% (8 out of 37) of the total approved drugs, showcasing a rapidly growing category among all new drug types.
With the rapid development of protein drugs, there is a heightened demand for advanced bioanalytical methods. Establishing an analysis method that simultaneously ensures specificity, precision, accuracy, and sensitivity, with high recovery rates and a broad linear range, is crucial. Such a method should be capable of simultaneously detecting and differentiating drugs, metabolites, and interfering substances. This analytical approach is essential to support pharmacokinetic and toxicokinetic studies of protein drugs, contributing significantly to the development of this class of pharmaceuticals.
Analysis Technology Development
Traditionally, ligand-binding assays (LBAs), represented by enzyme-linked immunosorbent assay (ELISA), have been the primary means for quantitatively analyzing antibodies and therapeutic protein drugs. However, LBAs require the preparation of specific antibodies, a time-consuming and costly process in the early stages of drug development. Moreover, LBAs cannot simultaneously measure drugs and metabolites, and the presence of antigen-binding domain metabolites may introduce errors in the test results. Additionally, different antibodies from various sources may exhibit significant differences in their reactions with the same protein. Interference from matrices and the presence of antidrug antibodies (ADAs) can also affect the accuracy of quantitative measurements by LBAs. Although these technologies based on the principle of antibody-ligand binding are highly sensitive, their limitations have spurred researchers to explore alternative detection platforms.
Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology for analyzing and detecting large-molecule protein drugs has gained increasing attention. In comparison to traditional LBAs, LC-MS/TS offers several crucial advantages:
- LC-MS/MS generally does not require the preparation of specific antibodies, allowing for method development in as little as a few days or weeks with lower development costs.
- LC-MS/MS exhibits higher specificity, resisting interference from target protein analogs. It can also detect protein degradation products and post-translational modifications (PTMs).
- LC-MS/MS has a broader linear range and is more easily transferable between different matrices and species.
The unique benefits of LC-MS/MS highlight its potential as a powerful alternative for the quantitative analysis of large-molecule protein drugs, overcoming some of the limitations associated with traditional ligand-binding assays.
Top-Down, Middle-Down, and Bottom-Up Approaches
Quantitative analysis of protein drugs using LC-MS/MS can generally be categorized into three proteomic methods: top-down, middle-down, and bottom-up.
Top-Down and Middle-Down Quantitative Analysis
Top-down and middle-down quantitative analysis methods employ high-resolution mass spectrometry (HRMS), such as ion trap mass spectrometry or time-of-flight mass spectrometry, for the quantitative analysis of intact and partially intact proteins. By eliminating protein unfolding and enzymatic digestion steps, direct mass spectrometric analysis is facilitated, expediting the bioanalytical workflow and theoretically providing more comprehensive biological information.
In top-down LC-MS/MS, complete proteins are quantitatively analyzed by selecting ions of intact proteins as precursor ions for collision-induced dissociation (CID). The appropriate fragment ions are then chosen for quantitative analysis in multiple reaction monitoring (MRM) mode. However, fragmenting intact protein segments with a molecular mass exceeding 10,000 is extremely challenging via CID. Additionally, due to the structural similarity of therapeutic protein drugs to endogenous IgG proteins, protein purification in the sample preprocessing stage necessitates the use of specific antibodies or ligands.
Middle-down methods for biological analysis of protein drugs include hinge region cleavage and deglycosylation. Hinge region cleavage involves specific enzymatic cleavage at the hinge region of antibodies, followed by antibody reduction, resulting in approximately 25,000 replicates of three subunits (Fc/2 fragments, Fab light chains, and Fab heavy chains, two of each). High-resolution mass spectrometry (HRMS) is then employed for quantitative analysis. Another method involves the use of N-glycanase F for deglycosylation of proteins, simplifying the protein's charge envelope to obtain a higher detection signal.
Furthermore, in pharmacokinetic studies, lower limits of quantitation (LLOQ), such as 0.1 ng/mL or lower, are often required for the study of in vivo drug metabolism and toxicity. Due to the limitations imposed by LLOQ requirements, top-down and middle-down quantitative analysis methods have not gained widespread use.
Bottom-Up Quantitative Analysis
Currently, one of the more commonly used methods for quantitative analysis of large-molecule protein drugs based on LC-MS/MS technology is the bottom-up approach, which quantifies at the peptide level. In comparison to top-down and middle-down approaches, quantitative detection at the peptide level can be achieved using low-resolution triple quadrupole mass spectrometers. The experimental workflow for bottom-up LC-MS/MS includes the selection of signature peptides, choice of internal standards (IS), optimization of LC-MS/MS conditions, sample preprocessing, optimization of enzymatic digestion conditions, and method validation.
The bottom-up approach offers advantages in terms of simplicity and sensitivity, making it a preferred choice for the quantitative analysis of large-molecule protein drugs using LC-MS/MS technology.
Schematics of top-down and bottom-up proteomics analysis (Neagu et al., 2022).
Practical Applications of LC-MS/MS
LC-MS/MS Method for Monoclonal Antibody Quantification
The use of monoclonal antibody (mAb) therapy is rapidly increasing. However, there are significant differences in mAb concentrations among individuals, potentially impacting exposure and treatment responses. This emphasizes the importance of monitoring mAb therapeutic drugs, necessitating the development of an effective and reliable method for accurate quantification in plasma samples.
Researchers have developed an efficient and accurate universal LC-MS/MS quantitative analysis method for the quantification of five monoclonal antibodies: bevacizumab, eculizumab, nivolumab, pembrolizumab, and trastuzumab. The LC-MS/MS method, utilizing trastuzumab as an internal standard, involves immunocapture purification of samples using protein G magnetic beads. A rapid trypsin digestion is performed on the magnetic beads at high temperature, followed by conventional pH elution. The analysis employs an Agilent 1290 ultra-high-performance liquid chromatography system with an AerisTMPEPTIDE XB-C18 column (2.1 mm × 100 mm, 1.7 μm) and an Agilent 6460 triple quadrupole mass spectrometer. The method demonstrates a linear range of 10 to 200 μg/mL.
Host Cell Protein (HCP) Analysis
Host Cell Proteins (HCPs) are process-related impurities derived from upstream processes in the manufacturing of biopharmaceuticals. Residual HCPs in drugs can affect product quality, stability, and safety. Therefore, developing a universal method to monitor residual HCPs is crucial.
Researchers have devised a sensitive and robust method capable of detecting residual HCPs down to 1 × 10-7. The process involves digestion at high protein concentrations, adding sodium deoxycholate in the reduction step to minimize HCP omission during digestion. Prior to LC-MS/MS analysis, a 50% acetonitrile wash in solid-phase extraction (SPE) is utilized to ensure monoclonal antibody removal. This workflow enhances the sensitivity of HCP identification by 10 to 100 times. The analysis utilizes an UltiMate 3000 RSLCnano liquid chromatography system with a CSH C18 column (75 μm × 50 cm, 1.7 μm) and an Orbitrap Exploris 480 mass spectrometer.
Reference
- Neagu, Anca-Narcisa, et al. "Applications of tandem mass spectrometry (MS/MS) in protein analysis for biomedical research." Molecules 27.8 (2022): 2411.
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