Amino acid analysis of peptide loading ratios in conjugate vaccines: a comparison of direct electrochemical detection and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate pre-column derivatization methods.
Amino acid analysis using direct electrochemical detection was compared with precolumn fluorescent derivatization using 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) for evaluation of the degree of covalent coupling of peptides to a carrier-protein complex derived from the bacteria Neisseria meningitidis. AQC derivatization was found to give superior sensitivity compared to electrochemical detection, with less interference from sample components such as carbohydrates or buffer salts. Hydrolysis time and temperature were optimized for maximal recoveries of the marker amino acid 6-aminohexanoic acid (epsilon-Ahx) and the unique amino acids S-dicarboxyethyl cysteine (SDCEC) and S-carboxymethyl homocysteine (SCHMC), which are generated upon the hydrolysis of the covalent linkage between the peptide and the carrier protein. Quantitation of these amino acids enabled the determination of the ratio of peptide to protein in the conjugate samples.
Nahas
Deborah D
DD
Merck Research Laboratories, 770 Sumneytown Pike, PO Box 4, WP16-107, West Point, Pennsylvania 19486, USA. debbie_nahas@merck.com
Palladino
Jennifer S
JS
Joyce
Joseph G
JG
Hepler
Robert W
RW
D000596:Amino Acids / Q000032:analysis*; D015119:Aminocaproic Acid; D000634:Aminoquinolines / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D002219:Carbamates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003545:Cysteine / Q000031:analogs & derivatives / Q000032:analysis; D004563:Electrochemistry / Q000379:methods*; D006710:Homocysteine / Q000031:analogs & derivatives / Q000032:analysis; D006868:Hydrolysis; D009345:Neisseria meningitidis / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D018074:Vaccines, Conjugate / Q000737:chemistry*
C078178:6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; D000596:Amino Acids; D000634:Aminoquinolines; D001426:Bacterial Proteins; D002219:Carbamates; D010455:Peptides; D018074:Vaccines, Conjugate; D006710:Homocysteine; D003545:Cysteine; D015119:Aminocaproic Acid