Two-Dimensional Liquid Chromatography Applications in Biopharmaceutical Analysis

Biopharmaceutical analysis typically involves the use of numerous, orthogonal, high-resolution analytical tools. As such, analytical characterization of biotherapeutic products is a resource-intensive activity and is in focus currently as biopharmaceutical manufacturers look to make development of these products cost-effective. In this context, two-dimensional liquid chromatography (2D-LC) has emerged as an efficient multidimensional approach for biopharmaceutical analysis. As proteins are inherently complex molecules, they offer significant analytical challenges owing to structural variations, post-translational modifications (PTMs), or the tendency to aggregate. Traditional one-dimensional liquid chromatography (1D-LC) methods have long been used to efficiently separate these biomolecules. However, over the past few years, 2D-LC has demonstrated superior separation capacity and resolution, garnering a spectrum of applications for entities such as monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), and other therapeutic proteins. Development of 2D-LC methods offers plentiful opportunities for innovation in instrumentation, automation, and data analysis. This article reviews literature related to 2D-LC applications in the biopharmaceutical field over the past six years (2018-2024).

Between January 2014 and July 2018, 155 new biopharmaceutical products were approved in the U.S., with monoclonal antibodies (mAbs) comprising the largest category at 44% of approvals (1) These biologics have their own biological, immunological, structural, and physicochemical complexity, which is a challenge for their quality assurance. Biotherapeutic products, specifically the complex ones such as mAbs, represent an increasingly fast-moving class of medicines that have 20–30 critical quality attributes (CQAs) that must be characterized (2). These CQAs include post-translational modifications (PTMs), product-related heterogeneities, process-related impurities, and host cell-derived contaminants. Further, structural heterogeneity of mAbs created by factors like charge variants, glycoforms, and other PTMs adds to the difficulty in their analysis (3,4). Liquid chromatography (LC) continues to be a key tool in this analysis (5) for assessment and monitoring of CQAs through all stages of the product lifecycle from manufacturing to expiry, ensuring efficacy, safety, and quality of the biotherapeutic product (6).

Conventional one-dimensional liquid chromatography (1D-LC) has been a gold standard in biopharmaceutical characterization for a variety of analyses over several decades. However, it offers limited chromatographic selectivity, lacks orthogonality in retention mechanisms, and suffers from low peak resolution and hence poor separation. Two-dimensional liquid chromatography (2D-LC), where the synergy of two orthogonal separation modes effectively solves these challenges effectively (7–9). For example, proteolytic digestion of large proteins may lead to the generation of >100 peptides, far exceeding the resolving power of any 1D-LC system with peak capacities of around 1000 or even of 10000 (10, 11). Use of 2D-LC has demonstrated how widely fractions such as peaks or peak slices can be selectively transferred between dimensions to highly increase the peak capacity of the system and resolve coeluted analytes (12). This facilitates accurate characterization of complex therapeutic products and supports manufacturing, development, and quality control (13). Coupling of 2D-LC with mass spectrometry (MS) is also becoming increasingly popular for applications that involve desalting, reduction, and digestion, followed by characterization using intact, reduced/middle, and bottom-up approaches (14,15). By employing orthogonal separation mechanisms, 2D-LC offers superior resolution, minimizes peak overlap, and enables the detection of low-abundance components within biopharmaceutical formulations. The versatility and capabilities of 2D-LC are rendered vital for the characterization of therapeutic proteins, the related components such as the bispecific antibodies and antibody-drug conjugates, host cell proteins, and excipients in drug formulations (Figure 1) (16).

Classification of 2D-LC Systems

The basic instrumentation of 2D-LC consists of the autosampler, binary or quaternary pumps, switching valves, two-column compartments, and two diode array detectors (DADs). The working principle of 2D-LC is controlled by pumps and loop valves among the two chromatographic chambers that resolve the heterogeneity of a biological sample using two distinct LC columns of different separation mechanisms. These resulting peaks can then be characterized in greater detail by employing MS (17,18). This allows the online analysis of samples without intermediate manual intervention, thereby reducing the disadvantages of offline cumbersome workflow such as buffer exchange and sample loss to contamination (19). Depending on whether the eluate transfers online from one dimension into another, 2D-LC separation systems exist with several combinations of these options. The term online defines a system in which the eluate from one dimension directly transfers to the next dimension, with the separation taking place in real-time in both dimensions (20,21). It implies faster analysis times, minimization of sample amounts needed, reduced matrix effects, increased analysis reliability, and high separation resolution of complex mixtures with fewer artifacts attributed to the method compared to traditional offline setups (22). Depending on what is being transported during the second dimension, 2D-LC methods can either be heart-cutting, high-resolution sampling (HiRes 2D-LC), or comprehensive (1D×2D). In the heart-cutting procedure, a main column (first dimension, 1D) first separates the constituents, and a modulator then selectively transfers selected fractions from the first column to a secondary column (second dimension, 2D), where further separation occurs (23). In contrast, in the comprehensive technique, the full eluate from the 1D step is passed through the 2D separation (24). HiRes 2D-LC is another mode intended for the separation of two closely eluting compounds, one being present at very low concentrations and hidden under the peak of another highly concentrated compound (25). Modern developments including a hybrid technique called multiple heartcutting (mLC–LC) draw significant attention, where only one or a few target compounds can be selected for characterization in the large detail of that particular peak(s) (26). Two principles govern the operations of 2D-LC: (1) the separation techniques must be orthogonal, and (2) along separation in other dimensions, the resolution of the first dimension must not be lost (27). There are several LC modes, including ion exchange chromatography (IEC), size exclusion chromatography (SEC), and reversed-phase liquid chromatography (RPLC), that can be combined and have found applications in the analysis of biopharmaceuticals. However, certain LC modes cannot be paired due to solubility and other compatibility issues (28). In the first dimension of 2D-LC, generally MS-incompatible mobile phases are employed, coupled with a fast-desalting step allowing for direct MS coupling without bias to chromatographic performance (29).

Given that biotherapeutics continue to be more complex, 2D-LC has cemented its place in modern analytical workflows (30, 31).

Applications of 2D-LC in Biopharmaceutical Analysis

The last decade has witnessed broadening of the biopharmaceutical pipelines to accommodate a spectrum of moieties than the traditional proteins such as monoclonal antibodies (mAbs) and many others like antibody-drug conjugates (ADCs), fusion proteins, and bispecific antibodies (BsAbs) (32). Advancements in the direct coupling of LC–MS-based multi-attribute monitoring (MAM) facilitate effective monitoring of various attributes in a single sample run workflow. Furthermore, 2D-LC–MS has been a powerful approach for characterization of therapeutic proteins because of its superior separation, orthogonality, and significantly higher resolution power (33).

Analysis of Critical Quality Attributes (CQAs) of mAbs

Monoclonal antibodies (mAbs) display great heterogeneity importantly size (aggregates and fragments) and charge variants arising during the production and purification process comprise post-translational modifications (PTMs) such as glycosylation, glycation, deamidation, oxidation, isomerization, and C-terminal lysine variation. Looking at the analysis of these crucial critical quality attributes (CQAs) of mAbs which are very essential in regulating the activity and function of proteins, a well-demonstrated place to do this is the 2D-LC platform (32). One-dimensional (1D) chromatography has a very difficult time due to co-elution problems, often requiring a lot of offline manual fractionation for further analysis, making 2D-LC steps a better alternative (34). Here are some of the major applications of the 2D-LC technique (Figure 2).

Charge and Size Heterogeneity of Monoclonal Antibodies

Researchers monitored low-abundance size and charge variants of an mAb in a single workflow using an innovative native two-dimensional size exclusion chromatography mass spectrometry/weak cation exchange chromatography (2D-SEC–MS/WCX–MS). In the 1D system, SEC was used for size variant separation, which separated high molecular weight (HMW) aggregates, monomers, and low molecular weight (LMW) fragments based on hydrodynamic radii (35). The eluted monomer fractions were transferred online to the second dimension (2D). While SEC–MS facilitated mass analysis of aggregates in the native form, WCX was operated in 2D for detection of acidic and basic charge variants. SEC and WCX eluents were directly coupled with MS, whereby SEC–MS confirmed the exact masses of monomers and aggregates, and WCX–MS provided intact mass profiles for each charge variant, confirming modifications (36). The 2D-LC method offered a shorter analysis time of 25 min as compared to stand-alone methods which required 90 min to analyze size and charge variants individually.

A 2D-LC workflow hyphenated to MS has been proposed for charge variant analysis of mAbs and biopharmaceutical proteins. It employed strong cation exchange chromatography (SCX) in the first dimension (1D) in which SCX resolved charge variants of mAbs and biopharmaceutical proteins (34). Reverse-phase liquid chromatography (RP-LC) was selected as 2D to desalt SCX fractions and facilitate mass-spectrometry compatibility. SCX-RP-LC was coupled to high-resolution Q-TOF-MS to successfully identify the major charge variants at the intact protein and subunit level (37). From the evaluated mobile phases for SCX, the MES/DAP buffer system combined with a sodium chloride gradient showed the best results for charge variant separation in combination with a non-porous sulfonated cation exchanger.

Researchers have described another approach for charge-based mAb heterogeneity characterization by 2D-LC. In this case, WCX was employed in 1D to separate acidic, basic, and neutral variants according to their net charge. The main peak was then transferred from CEX to 2D by the heart-cutting method for further resolution of the species. In 2D, anion-exchange chromatography (AEX) allowed elucidation of the coeluting species (38). The volatile-salt-based method minimized artificial artifacts by avoiding the desalting step and in-depth characterization through MS, unlike the traditional method. Thus, the proposed 2D-CEX−AEX method delivered further separation of the main peak of the CEX profile into its corresponding charge variants of mAbs which was not identified in the stand-alone CEX and AEX method.

A recent paper presented a novel method of 2D-LC mass spectrometry for monitoring monoclonal antibody (mAb) heterogeneity in cell culture harvest. It integrated Protein A chromatography (ProA) for titer estimation in the first dimension and cation exchange chromatography (CEX) for charge variant analysis in the second dimension. The use of volatile salts provided high-resolution direct infusion MS, which greatly differed from other methods (39). Using the heart-cut approach, the developed 2D-ProA-CEX–MS method successfully resolved titer and charge species like deamidation, isomerization, and succinimide intermediate peak in a quick 15 min runtime without any manual interference. The proposed 2D-ProA CEX– MS method offered superior performance and mass compatibility for titer and charge variant estimation of in-process, stability, variability, and biosimilar samples.

Post-Translational Modifications of Biotherapeutic Proteins

Besides mAb heterogeneity, PTMs can have profound effects on the structure, stability, and function of therapeutic proteins (40). Thus, their precise characterization is an absolute priority from the point of view of product quality and final performance.

In recent years, researchers have found that the 2D-IEC–MS was able to effectively identify oxidations at specific amino acid residues and quantify oxidized species. In the 1D, ion-exchange chromatography (IEC) or hydrophobic interaction chromatography (HIC) separated oxidized variants. Next, in the 2D, reversed-phase liquid chromatography (RPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF-MS) identifies oxidation sites at the peptide level (41,42). 2D-hydrophilic interaction chromatography (HILIC)–RPLC–MS has also allowed de-glycosylation profiling with a fine framework to resolve high-mannose and sialylated species. In the first dimension, glycoforms were enriched by affinity chromatography (Protein A) or HILIC while in the second dimension, RPLC–MS accurately defined glycan structures and site-specific modifications (42,43). A recently published paper demonstrated that with the help of 2D-IEC–RPLC–MS, asparagine deamidation is a post-translational modification that can be stressed to influence protein stabilization. In the first dimension, charge variants were sorted out employing IEC, with deamidation of the protein imparting a greater negative charge. The second dimension was then performed by employing the RPLC–MS, giving accurate information about the modification site at the peptide level (42).

Characterization of Antibody-Drug Conjugates (ADCs)

Antibody-drug conjugates (ADCs) are complex biopharmaceuticals composed of mAbs linked to cytotoxic payloads, and their intricate structure necessitates high-resolution analytical techniques for evaluating CQAs, drug-to-antibody ratio (DAR), and stability (44).

Experts emphasized the central role of 2D-LC in profiling ADCs, combining high-pH and low-pH RPLC with MS for enhanced resolution and analysis. The ADC proteins may undergo denaturation, reduction, alkylation, and enzymatic digestion to produce peptides that have been separated using high-pH (pH ~10) RPLC in 1D based on hydrophobicity. The peptides thus formed are blown off in the second dimension low-pH RPLC (pH ~2.5), which resolves charge and structural variants to aid in identifying impurities, conjugation sites, and glycoforms. In-depth analysis is performed using mass spectrometry (Q-TOF-MS) and matched with ADC-specific databases (44).

Researchers also implemented and developed a generic 2D-LC–MS strategy for the analysis of brentuximab vedotin, the gold-standard cysteine-linked ADC. The first dimension of this methodology relied on hydrophobic interaction chromatography (HIC) while the second one combined reversed-phase liquid chromatography (RPLC). 1D-HIC efficiently separated the ADC species according to DAR, while 2D-RPLC provided MS-compatible conditions for the identification of each DAR fragment. This represented an indirect identification of the HIC peaks leading to the quantification of unconjugated mAbs and the DAR distribution as well as a detailed analysis of ADC heterogeneity (45).

2D-LC thus eliminates the need for protein precipitation and provides information on both size variants and free drug-related species in a single analysis. SEC/RPLC 2D-LC can be used to identify the root cause of low recovery of the free linker/drug in ADC formulation. Different combinations of RPLC, HIC, and SEC in 2D-LC hyphenated with MS can successfully analyze different critical attributes of ADC and decipher key information on ADC structures.

Impurity Profiling

2D-LC with mass spectrometry, with its high separation power, is an effective analytical method for the accurate identification and quantification of even trace-level impurities, especially for the separation of host cell proteins (HCPs) in biopharmaceuticals (46).The technique addresses product consistency, patient safety, and regulatory guidelines established by regulatory bodies like the FDA and EMA by presenting a complete impurity profile needed for mAb production. Recently, experts have demonstrated that coupling high-pH and low-pH reversed-phase liquid chromatography (RPLC) with MS effectively improves the detection of HCPs and quantifies as low as 10 ppm. Biotherapeutic proteins (e.g. mAbs) in the sample are broken down by denaturation, reduction, alkylation, and digestion by trypsin to cleave them into peptides. Peptide fragments are then analyzed by 2D-LC (47) HCPs occur in trace levels (ppm levels) when highly concentrated mAbs are present. The first dimension utilizes high-pH (pH ~ 10) RPLC for the separation of peptides based on their hydrophobicity. Fractions are then concatenated into pools to improve protein coverage and sensitivity. In the second dimension, peptides are further separated by low-pH (pH ~2.5) RPLC, which enhances resolution and minimizes co-elution, allowing for more precise HCP identification. Separated peptides are then introduced into a high-resolution mass spectrometer, in which two scanning modes enable detection: full-scan MS at low energy, which detects intact peptide masses, and MS/MS fragmentation at high energy, which fragments peptides into constituent parts for exactly identified peptides against protein databases including the CHO cell proteome, to determine and quantify HCPs precisely (48).

Cell and Gene Therapy Products

A novel 2D-LC–MS platform has been recently proposed for high-throughput characterization of adeno-associated virus (AAV) capsids, which act as gene therapy vectors. In the first dimension, anion-exchange chromatography (AEX) effectively separates empty, partially filled, and full AAV capsids based on surface charge differences (48). The use of either tetramethylammonium chloride (TMAC) or tetraethylammonium chloride (TEAC) enhances resolution, delaying the elution of full capsids due to their higher negative charge from encapsulated DNA. The second dimension employs RPLC coupled with MS to resolve and quantify the three viral proteins (VP1, VP2, and VP3). RPLC-MS determines precise molecular weight, identifies protein variants, and detects post-translational modifications (PTMs), such as phosphorylation (+80 Da mass shift in VP1 and VP2), oxidation, and VP3 C-terminal truncation. This 2D-LC-MS platform is particularly well-suited for capsid integrity assessment, its composition analysis, and PTM profiling, making it a necessary tool for quality control and regulatory compliance in AAV-based gene therapy (50).

Challenges in 2D-LC Implementation for Characterization of Biotherapeutic Products

While 2D-LC offers significant advantages in biopharmaceutical analysis, it is important to acknowledge the challenges associated with its use. Solvent incompatibility between the two separation modes increased complexity due to large datasets analysis, and reproducibility concerns are key hurdles that must be addressed (51). Additionally, the sophisticated instrumentation required for 2D-LC is both complex and costly, which may limit its accessibility. Achieving consistent and reproducible results across multiple runs remains a concern due to the intricacies associated with the second-dimension separation. Also, the added second dimension contributes to increased overall run time, particularly if the second dimension entails slower separation (52).

Several solutions can be implemented to overcome these challenges posed by 2D-LC. Use of automated optimization software can decrease the complexity of method development, while a selection of orthogonal stationary phases would enhance the separation. Optimizing the sampling rates between dimensions, exploiting parallelism, and having a quick gradient elution can successfully control the prolonged run times (53). Problems with sample compatibility across dimensions can be solved by using active modulation of solvent to change mobile phase composition, matching appropriate stationary phase and solvent compatibility, and engaging dilution or evaporation methods to remove incompatible solvents before transferring analytes into the second dimension. Large file sizes from 2D-LC also can be easily interpreted with advanced and sophisticated visualization software (54).

Despite these obstacles, the prospects of 2D-LC in biopharmaceutical analysis remain bright and promising. Analysts are continuously developing innovative applications and refining analytical methods, even expanding to three- and four-dimensional chromatography with significantly higher resolution and analytical efficiency (39). With increasing acceptance, 2D-LC is solidifying its role as an indispensable tool in biopharmaceutical research.

Conclusion and Future Perspective

The implementation of 2D-LC in the development of biopharmaceutical products highlights its potential to streamline the analysis of biotherapeutics as a time and resource efficient approach that can deliver superior method attributes of sensitivity, selectivity, and resolution (52). Furthermore, 2D-LC hyphenated with mass spectrometry enhances analytical capabilities over traditional 1-D chromatography techniques by separating, detecting, and quantifying analytes in complex biological samples (8). It is evident that this technique is well-positioned to emerge as a key bioanalytical development in the time to come (53). The future of 2D-LC could include significant advancements such as: incorporation of artificial intelligence and streamlining of data processing to enable faster peak identification (54). Coupling 2D-LC with multiple detectors such as UV-vis, fluorescence, and refractive index detectors, allows for more comprehensive and precise screening of complex formulations. A fully automated 2D-LC system may further enhance its potential by enabling high-throughput analysis, reducing manual intervention, and accelerating applications in biopharmaceuticals (55), to facilitate real-time process monitoring for delivering batch consistency, regulatory compliance, and enhancing product quality (56).

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