Biochromatography

Reverse Phase HPLC and UPLC with various detectors (UV (PDA), FLD, RI CAD, ELSD, Electrochemical detectors (ECD))

Reverse phase HPLC is one of the most common methods of analyte separation and analysis. Separation of components of a mixture is achieved by taking advantage of their different solubility. Therefore, the partition of the solute between the mobile phase and the stationary phase provides the basis of the analysis. Common stationary phases used are C4, C8 and C18 derivatized silica. Typical mobile phase used with peptide and protein products is aqueous 0.1% trifluoroacetic acid and acetonitrile. Trifluoroacetic acid is a weak ion-pairing agent that also serves to maintain a low pH (pH ~2), thereby minimizing the ionic interactions between the product and the stationary phase. A gradient is usually used to separate the components, which typically consists of staring with ~2% acetonitrile and increasing the composition of the volatile mobile phase to about 98% acetonitrile. Typically, proteins elute at around 40-60% acetonitrile. If the product is amenable, formic acid can be used for the analysis could be monitored by mass spectrometry. These acidic conditions usually result in sharp narrow peaks. The flow is monitored at wavelengths (~210-220 nm), a region of the spectrum where the amide backbone absorbs.

Lancaster Labs uses primarily Waters HPLC/UPLC systems and Agilent HPLC and Fast HPLC Systems configured with various detectors including Refractive Index (RI) Detectors; Fluorescence Detectors (FLD), Photodiode Array Detectors (PDA); Electrochemical Detectors (ECD); Evaporative Light Scattering Detector (ELSD); Corona Charged Aerosol Detectors (CAD+); and Mass Spectrometric Detectors.

• Sample required: milligram to gram amounts of protein

Size-exclusion

Size exclusion chromatography (SEC) is a form of HPLC that separates molecules based on size. It fundamentally excludes large molecules from the pores of the stationary phase. SEC columns have published exclusion limits measured in Daltons and proteins whose molecular weights are larger than the exclusion limit are excluded and elute earlier than molecules than are captured by the pores. In ideal SEC, also called gel permeation chromatography, there is a linear relationship between the elution volume of a protein and the logarithm of its molecular size such that it is possible to estimate within a few percent the molecular weights for a specific protein as based off of molecular weight standards. One important note, this determination is based on the assumption that the protein is globular and symmetrical in shape.

Size-exclusion chromatography is generally performed to obtain a profile of the putative aggregates of the parent protein. Many proteins aggregate in the high salt content of human serum and the extent of which is measured by this method. Typically analyses of this type are run in high salt solutions (150 mM to 300 mM NaCl or other type) using beads/particles sizes of 5 µm to 10 µm. Denaturants such as guanidine or sodium dodecylsulfate (SDS) are sometimes added to eliminate aggregation and promote uniformity in protein shape (rod-like) for the purpose of enhancing separation of proteins. Furthermore the analyses are carried out at column temperatures of 4 deg C to 37 deg C with flows around 0.5 to 1 mL per min, monitoring the flow at a protein relevant wavelength.

• Sample required: milligram to gram amounts of protein

SEC-MALS

A special case of size-exclusion chromatography is one where the flow is monitored by a multi-angle light scattering detector (MALS). The method measures molecular weight (MW), hydrodynamic radius (Rh), and molar mass (MM) distribution of protein samples. In this analysis, the eluate is passed through a MALS detector (Wyatt Technologies) and the molecular radius of the solutes is determined by calculation of the laser refractions/reflections. This method works well with aggregated proteins, ranging in size from 100kDa to ~2000 kDa.

The method is capable of evaluating and comparing the degree of aggregation between samples. This method can be used to track and trend changes in aggregate profiles, but does not specifically identify and/or quantify individual aggregates or multimers. Moreover, with the addition of an RI detector, it is also capable of determining the percent mass attributed to glycans.

• Sample required: milligram to gram amounts of protein

Normal Phase

Normal phase chromatography is another form of HPLC. It is an adsorptive mechanism and is used for the analysis of solutes readily soluble in organic solvents, based on their polar differences such as amines, acids, metal complexes, etc. In normal phase chromatography, the most nonpolar compounds elute first and the most polar compounds elute last. The mobile phase typically are composed of nonpolar solvents such as hexane or heptane mixed with a slightly more polar solvent like isopropanol, ethyl acetate or chloroform. Longer retention times are achieved by increasing amount of nonpolar solvent in the mobile phase. Stationary phases are composed of silanols and functionalized silyl hydrides.
Typically the amount of the nonpolar component in the mobile phase must be 60% or greater with the exact point of increased retention depending on the solute and the organic component of the mobile phase. The aqueous component of the mobile phase usually contains from 0.1 to 0.5% formic or acetic acid, which is compatible with detector techniques that include mass spectral analysis.

Hydrophilk Interaction Liquid Chromatography (HILIC)

HILIC is a special case of normal phase chromatography. Normal phase chromatography is a type of liquid chromatography where the stationary phase is more polar than the mobile phase. The term normal phase used to distinguish this type of chromatography from reverse phase chromatography (this is most common type of analytical separation) where the stationary phase is less polar than the mobile phase.

• Sample required: milligram to gram amounts of protein

Ion Exchange Chromatography

Ion Exchange Chromatography is a form of HPLC. Ion exchange chromatography exists in two classes: anion (AEX) and cation exchange (CEX). This technique makes use of the fact that proteins are composed of positive, negative and neutral residues. Therefore, this analysis method separates according to overall charge of the surface of the protein. Anion exchange would have a positively charged stationary phase, such as polyamino (weak), diethylaminoethyl (moderate) and quaternary ammonium (strong). In contrast, cation exchange column would use negatively charged functionality, such as carboxymethyl (weak), organophosphate (moderate), and sulfo (strong) as means to retain/exchange the counterions. Both anion and cation ion exchange columns would have these functional groups tethered to a polystyrene bead. Mobile phase compositions are high salt concentrations with increasing ion strength and concentration. A benefit with ion exchange chromatography that may not exist in others forms is that biological activity is typically retained in this assay.

• Sample required: milligram to gram amounts of protein

Glycan Analysis

N-glycan analysis is performed on glycoprotein samples and can specifically determine the glycan content and identity (e.g. G0, G1, G2 content). Samples are treated with PNGase F to remove the N-linked glycans. These glycans are then labeled with 2AB and separated by normal phase HPLC with fluorescence detection.  A set of glycan standards (8 complex carbohydrates) are used to profile (i.e. identification and quantification) the sample.

• Sample required: microgram to milligram amounts of protein

Carbohydrate Determination

Carbohydrates such as D-(+)-mannose, D-(+)-glucose, N-acetyl-glucosamine, L-(-)-fucose, 2-deoxyglucose, D-(+)-galactose and N-acetyl-galactosamine are liberated from the glycoprotein or source of the carbohydrates via enzymatic or chemical means. The resulting carbohydrates are labeled with PMP (1-phenyl-3-methyl-5-pyrazolone) and analyzed by HPLC using reverse-phase separation and UV-detection. Chromatographic profiles are compared to commercially available carbohydrate standards that were also labeled with PMP.

• Sample required: microgram to milligram amounts of protein

Amino Acid Analysis

Amino Acid Analysis is performed according to Analysis #10030. The assay is carried out in two steps: 1) acid hydrolysis (usually hydrochloric acid) of protein into component amino acids using the validated Eldex Hydrolysis/Derivatization Workstation; 2) Derivatized the amino acids using the Waters AccQTag Chemistry. Eldex Hydrolysis/Derivatization Workstation Overview: This system is designed to provide convenient hydrolysis and pre-column derivatization as it applies to protein hydrolysis and amino acid modifications performed in the Biopharmaceutical Services area. Typically 6N hydrochloric acid at 110 deg C for 24 h is used to hydrolyze the protein and then it is removed under reduced pressure prior to derivatization. A Waters UPLC is used to separate the derivatized amino acids and from standard plots, the unknown amino acid concentration is quantified. We will evaluate the examine linearity, accuracy, and the matrix for interferences. We will report the amino acid composition in terms of number of residues, mol% or concentration of protein. One week of development will allow for two full runs of the analysis.

• Sample required: microgram to milligram amounts of protein

Extinction Coefficient Determination

The extinction co-efficient is a key analytical parameter from which many assays required the protein concentration. Typically, the client will provide the sequences of the product. The determination is carried out via AAA and the measurement of the absorbance at 280 nm. Beer's law will be used to determine the extinction coefficient.

A Nano-Drop Spectrophotometer or Molecular Devices M2 spectrophotometer are used in determining the absorbance at 280 nm.

Amino Acid Analysis is performed following Analysis #10030. Amino acid analysis is carried out in two steps: 1) acid hydrolysis (usually hydrochloric acid) of protein into component amino acids using the validated Eldex Hydrolysis/Derivatization Workstation; 2) Derivatized the amino acids using the Waters AccQTag Chemistry. Eldex Hydrolysis/Derivatization Workstation Overview: This system is designed to provide convenient hydrolysis and pre-column derivatization as it applies to protein hydrolysis and amino acid modifications performed in the Biopharmaceutical Services area. Typically 6N hydrochloric acid at 110 deg C for 24 h is used to hydrolyze the protein and then it is removed under reduced pressure prior to derivatization. A Waters UPLC is used to separate the derivatized amino acids and from standard plots, the unknown amino acid concentration is quantified.

• Sample required: microgram to milligram amounts of protein

Peptide Mapping

Peptide mapping of a protein or oligopeptide can be used for the purposes of identity or release. For example, in developing a trypsin based peptide map, the protein is contained in a volatile buffer solution such ammonium bicarbonate (100 mM) and treated with the enzyme at various enzyme:substrate ratios over a period of time (i.e. 5 to 24 h, typically). Other enzymes that have had applicability are Lys-C, Arg-N, Glu-C, and chymotrypsin. The digested mixture is then separated on a reverse phase U/HPLC system as depending on method requirements.

For each project, digest conditions are examined through reproducibility of the determined method of digestion and provide its impact on sequence coverage percentage (>95% targeted). MS analysis (LC-TOF or LC-MS/MS) is used to determine the sequence identities of key peptide fragments. We will examine the matrix for interferences.

• Sample required: microgram to milligram amounts of protein