Optimization of a trypsin-bioreactor coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs.
The optimization of a silica-based trypsin bioreactor and its use in the quality control of biotechnological drugs like peptides and proteins is described. Five bioreactors based on monolithic material have been prepared, with different amount of bound trypsin. The performances of these bioreactors were compared to the proteolytic activity of a bioreactor based on silica material. The trypsin-based chromatographic columns were coupled on-line with an LC/ESI/MS/MS system for digestion and identification of proteins. First, human serum albumin has been used as test protein to compare the ability of the bioreactors to hydrolyse high-molecular-weight proteins. The best chromatographic material (epoxy monolithic silica) and the optimum amount of enzyme bound (7.13 mg) have been identified to obtain the highest protein recovery and an analytical reproducibility of the whole digestion, separation and identification process. The optimized enzyme-reactor has been used for the on-line digestion of some biotechnological drugs such as somatotropin. Somatotropin for parentheral use has been analyzed, without sample pre-treatment, with both an on-line procedure and the traditional off-line procedure described in the European Pharmacopoeia. It was found that the cleavage efficiency (aminoacidic recovery, %AA) achieved within minutes by the developed protocol is at least comparable or even better than the conventional 4h consuming method.
Dipartimento di Chimica Farmaceutica, Università di Pavia, Via Taramelli 12, I-27100 Pavia, Italy.
C
C
Temporini
E
E
Perani
F
F
Mancini
M
M
Bartolini
E
E
Calleri
D
D
Lubda
G
G
Felix
V
V
Andrisano
G
G
Massolini
D000465:Algorithms; D000595:Amino Acid Sequence; D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D013006:Growth Hormone / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D004800:Enzymes, Immobilized; D011506:Proteins; D012709:Serum Albumin; D013006:Growth Hormone; D014357:Trypsin