Large Molecule HPLC

Large Molecule HPLC

Biomolecules are large, polymeric chemical compounds produced by living cells that serve as fundamental building blocks of living organisms and carry out many biological processes essential to life. The major biomolecule types include proteins, peptides, polynucleotides, carbohydrates, lipids, vitamins, and coenzymes. The nature of large molecule and biomolecule structure, their chemical diversity, the necessity to consider biological activity, and complex matrices demands an efficient characterization technique. Although there are many separation and analysis techniques available, high performance liquid chromatography (HPLC) is most commonly used. Due to their many functional groups and multiple conformations, HPLC biomolecule analysis is based on different modes such as reversed phase, size-exclusion, or ion exchange. Irrespective of the separation modes applied, efficient column packing and consistent stationary phase particle chemistry are critical for accurate and reliable biomolecule separation.   


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Reversed-phase biomolecule HPLC

Reversed-phase HPLC (RP-HPLC) is a sensitive and versatile technique used to separate and analyze proteins, protein fragments, and peptides. RP-HPLC uses a non-polar stationary phase and a polar mobile phase. Protein and peptide retention on the stationary phase follow adsorption and partitioning principles. Hydrophobic protein regions reversibly attach to the stationary phase. Proteins are eluted by increasing the non-polar nature of the mobile phase. Resolution can be affected by pore size, particle size, column length, and the hydrocarbon chain attached to the stationary phase.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography (SEC) is a non-denaturing chromatography mode that separates molecules by their size (i.e. hydrodynamic radius). This mode does not rely on analyte interaction with the stationary phase, but rather on random analyte flow through stationary phase particles. High molecular weight analytes elute earlier, as they are fully or partially excluded from stationary phase particle pores, while lower molecular weight analytes elute later, since they spend more time navigating the torturous path through the particle. SEC has been used for characterizing monoclonal antibody (mAbs) aggregates and fragments, estimating unknown protein molecular weights, and determining protein formulation stability.

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction chromatography (HIC) is a chromatography mode that separates analytes based on the degree of interaction between hydrophobic analyte moieties and hydrophobic stationary phase ligands. Due to their lower molecular weight and lower propensity for folding, HIC is usually not used in peptide separation. In high salt concentrations, protein hydration layers may be disrupted enough for hydrophobic surface regions to interface with the non-polar stationary phase. Salt selection is dictated by the Hofmeister series, which classifies cations and anions by their ability to disrupt protein hydration layers (chaotropic) or promote protein hydration layer formation (kosmotropic). Typical salts include ammonium sulfate, potassium sulfate, and sodium sulfate. Hydrophobic interaction chromatography is currently being used to determine the drug to antibody ratio (DAR) profile of antibody-drug conjugates (ADC).

Ion Exchange Chromatography (IEX)

Ion exchange chromatography (IEX) is a mode of chromatography that separates analytes by charge. Proteins and peptides are amphoteric, meaning they exhibit both acidic and basic functionalities. Acidic protein functionalities include aspartic acid, glutamic acid, cysteine, tyrosine, and the C-terminus α-carboxylate. Basic protein functionalities include arginine, histidine, lysine, and the N-terminus α-amine. Biotherapeutic charge variants can be detected and resolved by IEX. Charge variants can arise from messenger RNA (mRNA) transcript mistranslation and/or post-translational modifications, such as deamidation, oxidation, or glycosylation.

An IEX column must be selected based on the analyte isoelectric point (pI).  If the mobile phase pH phase is lower than the pI, the analyte will be positively charged and bind to a cation exchange column.  If the mobile phase pH is above the pI, the analyte will be negatively charged and bind to an anion exchange column.

Affinity Chromatography

Affinity chromatography relies on a specific interaction between an analyte and stationary phase ligand. Ideally, only the analyte of interest interfaces with the stationary phase, allowing all other sample components to pass through the column. A second mobile phase is then passed through the column to eluting the analyte.

Protein A chromatography is the most common form of affinity chromatography employed in the biopharmaceutical industry.  Protein A is a 42 kDa surface protein found in the cell wall of S. aureus.  This protein binds specifically to the heavy chain in the Fc region of IgGs, making this an ideal mechanism to separate IgGs from other sample components.  Most Protein A columns are manufactured by immobilizing the protein on a porous, organic particle.  However, monolithic format for Protein A chromatography has been produced, allowing for high sample throughput at various flow rates, without sacrificing efficiency.


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Calculate the saving in run time and solvent consumption when transferring a method from HPLC conditions to UHPLC conditions. Obtain guidance on change in back pressure as well as how to adapt injection volume and gradient conditions.

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