Biotechnol. Prog., 1999, Vol. 15, No.
4 3A
BIOTECHNOLOGY NOTABLES
Large-Scale Production of
Plasmid DNA
The development of process flow
sheets for the purification of super-
coiled plasmid DNA is reported. Using
a 4.8 kb plasmid, pMa5-L, as a model
system, it is demonstrated that the use
of RNase and high temperatures dur-
ing alkaline hydrolysis are unneces-
sary. Alternatively RNA was shown to
be completely removed by performing
clarification with a chaotropic salt and
precipitation with PEG-8000, followed
by ion-exchange and size exclusion
chromatography. Various chaotropic
salts were tested and shown to be
equally efficient in removing proteins
and nucleic acids from solution. Anion-
exchange HPLC was used to monitor
plasmid purity in the process streams.
Since the clarification and PEG concen-
tration steps were found to be respon-
sible for a significant decrease in over-
all yield, schemes which proceeded
directly from cell lysis to ion-exchange
and size exclusion chromatography
were investigated. The result is a final
downstream processing scheme which
obtained plasmid of comparable purity
and quality and which increased the
overall process yield by 38%.
Prazeres et al., Page 725
Intracellular pH Measurements
During Bioreactor Culture
A novel methodology is presented
which permits reliable and representa-
tive intracellular pH (pHi) measure-
ments on cells in a bioreactor environ-
ment. These measurements are carried
out under real cell culture conditions
rather than on cells resuspended in
buffers under nongrowth conditions as
reported previously. Hybridoma cells
were sampled during bioreactor culti-
vations and stained with the pH sensi-
tive dye BCECF-AM and analyzed by
flow cytometry. Experiments were per-
formed to determine the optimal condi-
tions to be used and the effects on the
measurements by such factors as cali-
bration, the presence of dead cells, the
duration of the conservation on ice, and
the duration of the resuspension of
stained cells.
The final procedure utilized a dye
concentration of 10 µM to stain a cell
concentration of 107 during an incuba-
tion time of 20 min. Supplementary
experiments were performed to demon-
strate that the pHi measured by this
methodology corresponds effectively to
the pHi of the cells in the culture at the
moment of sampling.
Cherlet et al., Page 630
Novel Microspheres with
Surface Amino Groups
Microspheres have been formed from
blends of poly(D,L-lactic-co-glycolic acid)
(PLGA) and poly(-CBZ- L -lysine)
(PCBZL) by a double-emulsification/
solvent evaporation technique. Micro-
sphere size was shown to be dependent
on both the PLGA/PCBZL ratio and
total polymer concentration. The pro-
duction of functional surface amino
groups by deprotection using either
acid hydrolysis or lithium/liquid ammo-
nia (Li0/NH3) reduction was evaluated.
Scanning electron microscopy was used
to assess microsphere surface morphol-
ogy. The capacity of the microspheres
to encapsulate a test solution of Texas
Red fluorescent dye was demonstrated
and used to further evaluate micro-
sphere integrity. From these studies,
the use of Li0/NH3 reduction for surface
deprotection was shown to be superior
to acid deprotection, leaving surface
morphology unaffected and absent of
any leakage of encapsulated solutions.
Hornsby and Zheng, Page 763
Spatial Biocompatibility in
Tissue Engineering
The spatial effects of nonwoven and
knitted polyester fabrics on cell aggre-
gate formation, proliferation, and func-
tion of human trophoblast ED27 cells
have been studied. Cells grown on 2-D
surfaces and knitted fabrics had faster
metabolic rates and higher prolifera-
tion activities as detected by cyclin B
assays. For nonwoven PET fibers, cells
grown in a low-porosity fibrous matrix
formed small aggregates (∼100 cells
per aggregate) while cells grown in a
high-porosity fibrous matrix formed
large aggregates (∼1000 cells per
aggregate). Such results were attrib-
uted to a critical porosity or fiber dis-
tance which controls cell spreading and
aggregation. The high-porosity matrix
had a relatively poor surface accessibil-
ity for cells to attach, spread, and pro-
liferate. A confocal laser scanning
microscope (CLSM) method was devel-
oped to evaluate the relationship of cell
proliferation to its surrounding archi-
tecture. Dual staining with propidium
iodide (PI) and 5-bromo-deoxyuridine
(BrdU) allowed cell imaging of prolifer-
ating and nonproliferating cells in the
cell fiber matrix within the same
sample. CLSM studies showed that
60% of cells in the small aggregates
found in the low-porosity matrix were
proliferating, while only 18% of cells in
the large aggregates found in the high-
porosity matrix were proliferating.
These results suggest that increasing
surface accessibility with lowered
matrix porosity would decrease aggre-
gate size and increase proliferation and
metabolic rates.
Yang et al., Page 715
Predicting Oscillatory Behavior
During Fermentation
Oscillatory behavior in continuous
fermentations of Zymomonas mobilis
has been examined. Extensive experi-
mental results are presented which
cover the complete range of dynamic
behavior: overdamped, underdamped,
and sustained oscillatory responses.
Experimental results confirm the inci-
dence of oscillatory behavior at com-
bined high feed substrate concentra-
tions and low dilution rates. A model
has been used to analyze the experi-
mental results and predict fermenta-
tion behavior. By comparing the esti-
mated models from the various
experimental runs, a pattern in the
instantaneous specific growth rate has
been identified in which the extent of
inhibition at low instantaneous ethanol
concentrations increases with
increased severity of the initial batch
fermentation (dictated by the initial
substrate concentration). Finally,
experimental evidence has indicated
the existence of periodic swings in cell
morphologies during sustained oscilla-
tory behavior. These observations sug-
gest that oscillations may be caused by
changes in the cell structure as the
cells adapt to changes in ethanol con-
centrations.
McLellan et al., Page 667
BP990285N