Author Gardner, Tara Conti
URN etd-1454132679612381
Title Delipidation Treatments for Large-Scale Protein Purification Processing
Degree PhD
Department Chemical Engineering
Advisory Committee
Advisor Name Title
William H. Velander Committee Chair
Richey M. Davis none
William L. Conger none
Keywords
* delipidation
* lipid removal
* protein purification
* transgenic milk
* sephadex
* TNBP
Date of Defense 1998-08-12
Availability unrestricted
Abstract
Triglycerides are the majority lipid component
of most biochemical mixtures and are virtually
water insoluble. Lipid removal is desired prior
to protein purification processing to decrease
nonspecific fouling of downstream
chromatographic matrices. Transgenic pig milk
was used as a model system to study
delipidation from therapeutic protein sources.
The majority of triglycerides was extracted
from stable lipid micelles and removed with a
method that can be incorporated in
downstream protein purification processing
without denaturing the target protein. An
efficient delipidation treatment used TNBP, a
non-polar solvent, to extract lipid micelles and
then phase transfer milk lipids into a
TNBP-swelled dextran particulate. A batch
incubation of a whey/TNBP mixture with
pre-swollen Sephadex LH-20 or
hydroxyalkoxypropyl dextran (HAPD) beads
at 4 C for 24 hours removed 67 + 2 %
(0.645 mg triglycerides/ml Sephadex LH-20)
and 71 o + 1 % (0.628 mg triglycerides/ml
HAPD) of the triglycerides present in the
skimmed transgenic whey, respectively. Fully
swollen beads removed 20% more
triglycerides than beads which were wetted
but not swollen in TNBP, indicating that a
larger phase volume and internal adsorption of
the lipids onto the Sephadex matrix dominates
over surface adsorption. Polyclonal ELISAs
indicated that 89 + 6% of the recombinant
human Protein C was still present in the
transgenic whey after this delipidation
treatment, indicating this treatment did not
denature or harm the target protein.
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