Effects of polyethylene glycol molecular weight and concentration on lactate dehydrogenase activity in solution and after freeze-thawing.
Lactate dehydrogenase (LDH) is a tetrameric enzyme that has been used as a model for labile protein drugs. Polyethylene glycols (PEGs) have been proposed as excipients to stabilize labile proteins in solution and during the freezing portion of the lyophilization cycle. The aim of this study was to determine the effects of PEG molecular weight and concentration on the activity of LDH. In general, PEG protection increases with PEG molecular weight and concentration. PEGs slow the loss of activity in LDH solutions stored at 4 degrees C, but are not sufficiently effective to allow for a solution product. PEGs 8000, 10,000, and 20,000 show full freezing protection at less than 0.01%, while lower molecular weight PEGs need higher concentrations to produce protection upon freezing. Circular dichroism (CD) studies of LDH solutions before and after freezing with PEG 400 and PEG 8000 confirm the activity studies. The CD spectrum of LDH before freezing shows the classic alpha helix pattern. After unprotected LDH solution is frozen and thawed, the CD spectrum erodes. Low concentrations of PEG 8000 (1% or less) preserve the alpha helix profile after freezing of the samples. PEG 400 preserves the alpha helix CD profile in a stepwise fashion with increasing concentrations. The CD and activity data suggest that PEGs can protect alpha helix structures and activity of LDH through the freezing process.
Mi
Yanli
Y
Parenteral Medication Laboratories, University of Tennessee, College of Pharmacy, Memphis, Tennessee, USA. ymi@utmem.edu
Wood
George
G
Thoma
Laura
L
Rashed
Sahar
S
D000818:Animals; D002942:Circular Dichroism; D005079:Excipients; D005615:Freezing; D007770:L-Lactate Dehydrogenase / Q000097:blood*; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011817:Rabbits; D012996:Solutions; D013048:Specimen Handling; D017550:Spectroscopy, Fourier Transform Infrared
D005079:Excipients; D012996:Solutions; D011092:Polyethylene Glycols; D007770:L-Lactate Dehydrogenase