Purpose

Samples of purified proteins and monoclonal antibody proteins can be hydrolyzed to release the bound amino acids to accurately determine a concentration of each individual residue. A performic acid pretreatment on the protein ensures that the sulfur containing amino acids Cysteine and Methionine are not broken during the hydrolysis procedure. To preserve any Tryptophan, samples are put through an alkaline hydrolysis. This method is for accurate determination and quantification of amino acids in monoclonal antibodies.

Summary

Samples are digested using an acid-stable hydrolysis procedure to release the bound amino acids present in the protein or monoclonal antibody. An acid-stable amino acid internal standard of Norvaline is added prior to hydrolysis to accurately compensate for any residues that may have been destroyed during sample digestion. Samples are also put through a performic acid pretreatment to add a sulfone protecting group on Cysteine and Methionine present in the sample, generating Cysteic Acid and Methionine Sulfone before applying an acid hydrolysis procedure. Samples are also prepared using an alkaline hydrolysis method to generate Tryptophan. Samples are then diluted to an appropriate concentration based on the protein content of the sample. For the samples having undergone acid-stable hydrolysis and performic acid pretreatment hydrolysis, a solution of a deproteinizing solution (5-sulfosalicylic acid hydrate), internal standard (L-2-Amino-3-Guanidinopropionic Acid Hydrochloride), and sodium buffer is added to the sample to achieve the target dilution. For samples having undergone alkaline hydrolysis, a solution of phosphate buffer is added to the sample to achieve the target dilution. All sample mixtures are then filtered into an HPLC vial.

Hydrolyzed amino acid analysis (acid-stable and performic acid pretreatment hydrolyzed samples only) is performed on amino acid analyzers. Amino acids are separated by cation exchange chromatography using various sodium eluents. Absorbance is measured at 440 and 570 nm following post-column color development by Ninhydrin reagent. Appropriate standards are run to ensure each sequence is running properly and used in the quantification of samples using peak areas and applying an external standard technique.

Alkaline hydrolyzed tryptophan amino acid analysis is performed using a reversed phase C-18 column with an aqueous buffer containing organic solvents by HPLC.  Peak detection uses a combination of in-line, programmable, and scanning fluorescence detectors. Appropriate standards are run to ensure each sequence is running properly and used in the quantification of samples using peak areas and applying an external standard technique utilizing a calibration curve.

Bovine serum albumin was used to validate this methodology, as it is readily studied and has known amino acid residue values. Using the molecular weight of the monoclonal antibody, the total number of each amino acid residue is calculated. For monoclonal antibodies with an unknown molecular weight, bovine serum albumin values are used to calculate the residue values of the protein.

Amino Acids Quantified
Cyanocobalamin (B12)Riboflavin (B2)

Hydroxyproline

Glutamic Acid

Phenylalanine

Cysteine

Aspartic Acid

Proline

Isoleucine

Lysine

Methionine

Threonine

Glycine

Leucine

Histidine

Tryptophan

Serine

Alanine

Valine

Tyrosine

Arginine