Biotechnology
Pharmaceutical Proteins
Complex, highly glycosylated proteins of pharmaceutical interest, which are derived from mammalian cell culture, were investigated for their heat resistance in a number of buffers, differing in salt composition, pH-value and conductivity.
Recombinant tissue plasminogen activator (tPA, IEP 5.0-8.0) is a 65 kD glycoprotein with proteolytic activity.
Humanized monoclonal antibodies (huMAb1, IEP 7.0-7.6, huMAb2, IEP 8.8-9.3 and huMAb3, IEP 7.8-8.5), glycosylated, MW 160 kD, were evaluated accordingly. Differential Scanning Calorimetry (DSC) showed a melting temperature (TM) for tPA at 69.9oC, a TM1 for a modified tPA variant at 64.2oC, a TM1 for huMAb1 at about 64.6oC, a TM1 for huMAb2 at about 71.6oC and a TM1 for huMAb3 at about 65.7oC. The exposure time in DSC is in a minute/oC-range and hence exceeds the exposure time to microwave heating by about 3 log
tPA could be processed at temperatures up to 120oC while maintaining its monomeric state and full biological activity, i.e. fibrin binding and plasminogen activation. The tPA variant molecule which was already shown to be more heat sensitive in the DSC also tends to aggregate at a lower temperature than the native tPA.
At temperatures of >98oC even highly resistant viruses such as porcine Parvovirus (PPV) are entirely inactivated.
HTST Treatment of Immunoglobulins
Different buffer salts,
salt molarities and pH-values were investigated with respect to their
influence on protein solubility. The buffer components were selected
regarding large scale manufacturing. Citrate buffer was identified to be
superior to phosphate buffered saline (PBS), and provides a simple,
inexpensive buffer system which significantly pronounced the recovery of
protein monomers; a slightly acidic pH was favorable.
At a recovery of > 95% monomers, the biological activity of both molecules was found to be indistinguishable from non-treated material in a cell-bound analytical assay.
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©1999 Charm Bioengineering, Inc.