LC-MS-Based Protein Therapeutics Analysis

Thermo Scientific offers both anion- and cation-exchange columns in the MAbPac and ProPac column families described on Charge Variant Analysis HPLC Columns. Among these, our ProPac 3R SCX and ProPac 3R SAX Columns offer the most advanced column technology for charge-variant analysis in our portfolio. Using a novel monodispersed polymeric resin, ProPac 3R IEX Columns deliver the resolution power, lot-to-lot reproducibility, and robustness needed for reliable charge-variant analysis workflows.

Thermo Scientific CX-1 pH Gradient Buffer Solutions can be used to generate highly reproducible, linear pH gradients using cation-exchange chromatography, as described in our CX-1 gradient buffers product specification sheet, Simple method development for charge variant characterization.

Proteins therapy products can be subjected to a variety of biochemical modifications during processing, delivery, and storage, which have been shown to affect the safety and efficacy of these therapeutics.

Oxidation of amino acid residues can alter the hydrophobic nature of a therapeutic protein either by increasing the polarity of the amino acid’s oxidized form or by causing a conformational change of the therapeutic protein.

Although conformational variance analysis can be accomplished through charge variance analysis, hydrophobicity-based HPLC methods such as reversed phase liquid chromatography and hydrophobic interaction liquid chromatography (HIC) have proven to be the most suitable solution for separating protein variants with little difference in hydrophobicity, such as oxidation variants.

Oxidation that causes the conformational variance can come from interactions with surfactants in the production process or from other points in the process. As a result, there is an incentive to monitor the surfactants used throughout production to discover the root cause of any non-conformities related to drug stability. A high-resolution surfactant analysis of polysorbate, a commonly-used surfactant in biopharmaceutical manufacturing, by charged aerosol detection (CAD) can be found in the application note Polysorbate 80 profiling by HPLC with mass and charged aerosol detection.

Thermo Scientific MAbPac HIC-20 Columns are high-resolution, silica-based HIC columns with a unique proprietary stationary phase that provides high resolution and rugged stability and is particularly good for antibody (AB) oxidation and analysis of disulphide protein variants. Separation of mAb oxidation variants using a MAbPac HIC-20 column is further described in the application note Separation of Monoclonal Antibody (mAb) Oxidation Variants on a High-Resolution HIC Column.

Antibody-drug conjugates (ADCs) are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer and typically composed of a mAb covalently attached to a cytotoxic drug via a chemical linker. ADCs are a hot topic in research and development of anticancer drugs as they combine the advantages of highly specific targeting abilities with highly potent killing effect to achieve accurate and efficient elimination of cancer cells.

The conjugation of drugs often results in ADC molecules which are heterogeneous with respect to both the distribution and loading of cytotoxic drugs on the mAb. Unconjugated mAbs have significantly lower potency, and the ADCs with high drug loads are subject to rapid renal clearance.

As the number of drugs attached to the mAb has been shown to directly affect the safety and efficacy of the drug, it is critical to fully characterize and monitor the heterogeneity of ADCs during development and production.

The attachment of a cytotoxin alters the hydrophobicity of the antibody so that, during HPLC, the least hydrophobic unconjugated antibody will elute first, and elution time increases as the number of drugs attached increases. HIC is, therefore, considered to be the method of choice for characterizing the distribution of ADC molecules with different drug-to-antibody ratios (DARs) – more information on this topic can be found in the application note High-Resolution Separation of Cysteine-Linked Antibody-Drug Conjugate Mimics Using Hydrophobic Interaction Chromatography. Thermo Scientific MAbPac HIC-Butyl Columns are packed with C4-bonded polymeric particles and the hydrophilic nature of polymer particles and optimal density of butyl functional groups lead to excellent biocompatibility, low carryover, and high resolution.

Mass spectrometry (MS) is also becoming a popular method of ADC characterization as MS provides a more in-depth understanding of ADC biomolecules and their various species. For MS applications, it is important to use MS-suitable solvents, which rules out the use of HIC columns and their high salt content. An alternative column that offers MS-compatibility is the Thermo Scientific MAbPac Reversed Phase (MAbPac RP) Column, which contains a polymer particle with a significantly large pore size (1500 Å) that allows proteins to diffuse very efficiently. The polymer sorbent is an effective interaction source for hydrophobicity that also makes the column very robust, allowing for the use of stronger organic solvents, effective column cleaning, and reduced carryover. The paper Development of NISTmAb-derived Antibody-drug Conjugate (ADC) Standards provides an example of a reversed phase, MS-based, NISTmAb-derived ADC standard application protocol.

Glycosylation – the attachment of sugar moieties to proteins – is done as part of the post-translational modification (PTM) process to optimize a biotherapeutic’s efficacy. The PTM is characterized by various glycosidic linkages, including N-, O-, and C-linked glycosylation, glypiation (GPI anchor attachment), and phosphor-glycosylation.

Glycosylation is one of the key critical quality attributes (CQAs) of mAb-based biotherapeutics, as any structural changes can impact a biological drug’s safety, efficacy, clearance, and immunogenicity.

Accurate and precise quantification of mAb-released N-glycans is best performed on a hydrophilic interaction liquid chromatography (HILIC) column due to the polarity of the glycans. Thermo Scientific Accucore 150 Amide HILIC UHPLC Columns have a unique proprietary selectivity bound to solid core particles, which increases overall efficiency of the column without substantially increasing backpressure. For more in depth explanation of solid core technology, visit our Accucore HPLC and UHPLC Columns webpage. Accucore 150 Amide HILIC columns have a larger pore size of 150 Å that is more compatible with peptides, allowing for improved peak shape and increased separation accuracy. The application note Accurate and precise quantification of mAb-released N-glycans with an amide HILIC column describes the effectiveness of the quantification of human IgG-released N-glycans when using an Accucore 150 Amide HILIC column.

Due to the inherent complexity of biotherapeutics, regulatory agencies require the comprehensive characterization of these drug product to ensure product quality, safety, and efficacy. Primary sequence verification and the identification and relative quantitation of post-translational modifications (PTMs) of proteins are important characterization steps for therapeutic proteins that are frequently performed using peptide mapping methods.

Peptide mapping is used to measure several critical quality attributes (CQAs) required for the characterization of any biotherapeutic proteins and involves the treatment of proteins with a protease (e.g., trypsin) to produce a series of peptides which are separated, detected, and analyzed by liquid chromatography mass spectrometry (LC-MS). Liquid chromatography-mass spectrometry and the related multi-attribute method (MAM) analysis enable characterization and monitoring of a wide range of CQAs simultaneously as described in the article Optimized Multi-Attribute Method Workflow Addressing Missed Cleavages and Chromatographic Tailing/Carry-Over of Hydrophobic Peptides.

Host cell proteins (HCPs), which are process-related impurities derived from Chinese hamster ovary (CHO) cell host organisms during biotherapeutic manufacturing, can also be analyzed through peptide mapping with NISTmAb verification of common CHO peptides using an UHPLC-MS application setup.

Various Thermo Scientific columns can be used for peptide mapping. Thermo Scientific Acclaim VANQUISH C18 UHPLC Columns are highly retentive and contain 2.2 µm silica particles with a pore size of 120 Å. These columns also have a surface area of 300 m²/g and 18% carbon content, ensuring maximum retention of complicated samples such as for peptide mapping.

Thermo Scientific Hypersil GOLD C18 Columns are also excellent for peptide mapping analysis as they are packed with ultrapure silica particles bonded to short butyl chains for weak hydrophobic retention. The quality of the silica minimizes secondary interactions that could cause undesired retention such as peak tailing. Hypersil GOLD C18 has a pore size of 175 Å, which allows for continuous diffusion and less resistance to mass transfer and is highly beneficial for the peak shape of the peptide.

Regardless of which column you choose, Thermo Scientific SMART Digest Trypsin Kits provide a significant advance in sample preparation for biopharmaceutical protein research. The kits provide a method for fast and simple protein digestion with high reproducibility, high sensitivity, and high levels of data quality in a format that is compatible with automation, reducing the time spent on a digest to as little as 45 minutes. Visit our Protein Digestion for HPLC and MS Peptide Mapping webpage for more information.