Characterization/Identity

CE-IEF

Isoelectric Focusing (IEF) is a characteristics method for the identification and differentiation of proteins. It separates the protein according to their isoelectric points (pI). Proteins are amphoteric molecules; they carry either positive, negative or zero net charge, depending on the pH of their surrounding. When a mixture of proteins is applied at a point in a pH gradient, the different proteins have a different net charge at this pH value. The positively charged proteins migrate towards the cathode, the negatively charged towards the anode, until they reach the pH value, where they are isoelectric. In cIEF technique, protein samples are introduced into a capillary with a mixture of ampholyte buffers which are a polymerized mixture of monomers that contain weakly acidic and weakly basic groups. By using an acidic solution on the anode side of the capillary and a basic solution on the cathode side and applying electric field, the ampholytes molecules orient themselves with respect to their pKa value, generating a pH gradient within the capillary. Proteins migrate through the ampholyte mixture and focus at a pH where they become electrically neutral (pI). After focusing, the zones must be mobilized to the detection window by applying pressure together with focusing potential.

Resolution of pI species on samples is performed by Capillary Isoelectric focusing (cIEF) using a ProteomeLab PA 800 Capillary electrophoresis system. An applicable pI range of 3-10 will be used. Separation is performed on a neutral capillary filled with Ampholyte/sample mixture with a 15 kV focusing voltage applied over 6 minutes. The sample is then mobilized to the detection window at 21kV and 0.5 psi presure. The sample (main peak) and impurities (if any) are detected at 280 nm. The migration time of the sample is correlated with the calibration plot of pI vs the migration time of the pI markers.

• Sample required: 100µg -500µg protein

IEF - GEL

Isoelectric Focusing (IEF) is a characteristic method for the identification and differentiation of proteins. It separates the protein according to their isoelectric points (pI). Proteins are amphoteric molecules; they carry either positive, negative or zero net charge, depending on the pH of their surrounding. When a mixture of proteins is applied at a point in a pH gradient, the different proteins have a different net charge at this pH value. The positively charged proteins migrate towards the cathode, the negatively charged towards the anode, until they reach the pH value, where they are isoelectric.

Samples are loaded at different dilutions (1:1/1:10) on to a Serva Precotes pH 3-10 gel along with IEF markers pH 3-10. These are then subjected to electrophoresis using a Multiphor II system.. The gel is stained with either Serva Violet or Colloidal blue, destained and scanned using a densitometer (Molecular dynamics or BIO-RAD GS-800).

• Sample required: 100µg-500µg protein

Western Blot

Blotting is the transfer of large molecules on to the surface of an immobilizing membrane. The western blot (protein immunoblot) is an analytical technique used to detect specific proteins in a given sample (i.e. drug product). This blotting technique is used to establish protein identity and purity in respect to host cell protein. Native or denatured proteins are separated by gel electrophoresis (Novex X Cell II system). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (identified) using antibodies specific to the target protein. After the proteins are transferred to the membrane, the membrane is incubated in series of steps - Incubation with a blocking agent to bind non-specific protein binding sites; Incubation with an antibody with specificity to bind to the protein; Incubation with an alkaline phosphatase secondary IgG to bind with the specific antibody; Incubation with a substrate for alkaline phosphatase to develop color at the site of the protein-antibody complex on the membrane.

• Sample required: 100µg-500µg protein

Carbamylation

Carbamylation is the addition of urea to a lysine residue (+44 Da). This chemical reaction occurs in development of the products isolation and purification process. To determine the location and extent of the modification, a peptide map is performed on the LTQ-Orbitrap, TSQ Vantage Triple Quad or Agilent TOF. Digest conditions are examined through reproducibility of the determined method of digestion and provide its impact on carbamylation and other possible modifications to the protein. MS/MS analysis is used to determine the sequence identities of key peptide fragments. Matrix interferences (i.e. aluminum content) are also examined.

• Sample required: microgram to milligram amounts of protein

Deamidation

Deamidation is the hydrolysis the asparagine and glutamine side chain amides, of a peptide or protein, releasing ammonia. Overall, the mass difference of on deamidation is 1 Da. This chemical reaction occurs through direct aging of the protein in appropriate pH conditions and possibly in the development of the purification process. To determine the location and extent of the modification, a peptide map is performed. Digest conditions are examined through reproducibility of the determined method of digestion and provide its impact on carbamylation and other possible modifications to the protein. MS/MS analysis is used to determine the sequence identities of key peptide fragments. Matrix interferences (i.e. aluminum content) are also examined.

• Sample required: microgram to milligram 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

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

Free Thiol Determination

The amount of free sulfhydryls/thiols present on a protein can be determined. Proteins contain cysteine residues, which have the ability to form disulfide bonds. The disulfide bonds restrict the molecule and force the protein/peptide into conformationally restricted forms. This test is intended for the quantification of any free thiols groups present on the molecule. The principle of the test is a chemical reaction occurs between the protein and the test reagent, Ellman’s reagent. The assay is carried out in a Molecular Devices M2 plate/cuvette reader where the absorbance at ~410 nm is recorded. Standards (i.e. cysteine, glutathione) are used to relate thiol content to the sample. Calculations as based off of the direct absorbance reading for the sample encompassing the standard curve reveal the molar amount of the free thiol in the sample. This value is then related to the protein molar amount.

• 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

N, C-Terminal Sequencing

MS/MS analysis on the LTQ-Orbitrap is performed to determine the N-terminal sequence identities of key peptide fragments. In cases where the sequence is known, a suitable protease will be used to cleave the N-terminal amino acids (~15-25 residues). Sequencing will be performed on the LTQ-Orbitrap. For the determination of N-terminal sequence, additional protease digests (peptide maps) would be required to show overlap with initial protease cleavage site. Manual alignment of the peptides would then be possible.

• Sample required: microgram to milligram amounts of protein

MS Oxidation Assay

A method for the determination of oxidation extent of a protein has been employed. The method examines the LC chromatogram of a protease digested sample with UV/MS-TOF detection. A direct comparison of the oxidized peptide with the native peptide is examined. The native peptide is identified by either MS confirmation or through authentic material if needed. Alternately, the oxidized peptide is identified in the chromatogram and analyzed by LC-MS/MS (MRM) on a triple quadrapole mass spectrometer.

• Sample required: microgram to milligram amounts of protein