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					                                                            Sam Maurer                   Group B
                                                            10.28 Cell Culture Lab Assignment #1
1

J. R. Zabrecky and W. Sawlivich. Methods. 32: 3-6 (2004).
―Purification of the heat-shock protein, Gp96, from natural sources.‖

        Due to the heat-sensitive nature of the glycoprotein Gp96 and relative instability of its
disulfide linkages, it is necessary to purify it at a low temperature, as quickly as possible, to
prevent degradation. Zabrecky and Sawlivich have adapted such a purification process from
Srivastava (1986), operating on a scale such that .2 – 80 mg of Gp96 can be produced from 5 –
1000 g of starting tissue or cell pellet. The procedure should be particularly attractive a
laboratory course, as it can be completed in the course of a single day. The process begins with
an optional ammonium sulfate precipitation step, the omission of which can double or triple the
yield of Gp96 at the cost of reducing purity by 15%. Following this, the filtrate of the
precipitation reaction is subsequently resolved via Con A affinity and DEAE anion exchange
chromatography. The final purified product has been found to be active in both in vitro and in
vivo biological assays. The authors note that this product should be stored at -80 oC to prevent
degradation and take the precaution of adding appropriate protease inhibitors to the reaction
mixture, supplying further evidence that Gp96 is a somewhat unstable protein. As the procedure
described takes 6 – 7 hours to complete fully even on a small scale (<50 g tissue), it is crucial to
avoid contamination and operate at low temperature with this sensitive material.

A. M. Feldweg and P. K. Srivastava. Intl. J. Cancer: 63: 310-314 (1995).
―Molecular heterogeneity of tumor rejection antigen heat-shock protein Gp96.‖

Certain glycoproteins of molecular weight 96 kDa, collectively referred to as Gp96, have been
shown to mediate tumor-specific immunogenicity of MethA mouse cancer cells when given in
restricted dosages. As a result, its application as a possible vaccine against certain tumors is
currently being studied. In this article, Srivastava and Feldweg have explored the purification of
Gp96 and characterized several distinct immunogenic heat-shock proteins of molecular weight
96 kDa. After preparing a sample of Gp96 on a silver-stained gel, researchers discovered that it
resolved on a silver-stained gel as three to four distinct bands. Confoundingly, each of these
bands was found to bond to a known anti-grp94 monoclonal antibody. Ultimately, it was
discovered that the bands consisted of Grp96 glycoproteins that shared the same peptide
sequence and glycosylation, but differed in conformation and degree of phosphorylation. As a
result, each of these 96-kDa glycoproteins indeed bonded to the monoclonal antibody, but did so
at a different binding site. Additionally, a group of more slowly-migrating 110-kDa protein was
discovered and purified. The authors suggested that these proteins co-purify with the Gp96
glycoproteins and may actually be responsible for the tumor-specific immunogenicity of MethA
cancer cells rather than Gp96 itself.
L. Chu and D. K. Robinson. Current Opinion in Biotechnology. 12: 180-187 (2001).
―Industrial choices for protein production by large-scale cell culture.‖

         In this review article, Chu and Robinson explore recent advances in the application of
stirred tank reactors to mammalian cell culture. Specifically, they contrast suspension cell-
culture processes with adherent cell-culture processes. Suspension cell-culture processes have
become more feasible since the development of polymeric shear-reducing additives, such as
Pluronic F68. As suspension processes have also been proven to be suitable to scale-up without
diminishing product quality, they have become the standard for large-scale industrial
applications. The other major limitation of suspension processes, waste removal and
replenishment of fresh medium, has been solved somewhat by perfusion culturing, which is
uniquely suited to protein production applications. Adherent cell-culture processes present the
opposite challenges—while it is facile to change the media and mix the solution without
damaging the adhered cells, scale-up is difficult because of the necessity that all cells adhere to
some surface of the reactor. As a result, industries operating large-scale adherent cell culture
processes have adopted ―roller-bottle,‖ ―stacked-disk,‖ or ―microcarrier bead‖ bioreactor designs
with high surface-area-to-volume ratios. A major limitation common to both processes is the
accumulation of dissolved CO2, as stirred tank reactors dissolve gases efficiently with minimal
aeration. In response to this, the authors cite a paper by Pattison et al. in which nitrogen
sparging and an online CO2 analyzer were used together to control its concentration to a
reasonable level. Citing recent industrial statistics, authors make the ultimate conclusion that
stirred tank bioreactors are the preferred instrument for large-scale cell-culture processes
producing recombinant proteins and antibodies.
                                                           Sam Maurer                   Group B
                                                           10.28 Cell Culture Lab Assignment #1
2

Calculated kLa values and plots appear on the following page.

From the time vs. dissolved oxygen plot attached, it appears that the solution in the wave reactor
becomes saturated with dissolved oxygen more quickly when the rocking angle and rocks per
minute (rpm) are increased. An increase in rpm affects the saturation rate more than an increase
the rocking angle. The difference in rate is not noticeable below a dissolved oxygen
concentration of 20%. Had more time data points been sampled for each rocking setting, it also
might have been possible to determine whether the maximum dissolved oxygen concentration
varies as a function of rpm or rocking angle. In general, the dissolved oxygen concentration
increases more rapidly at higher concentrations.
                                                            Sam Maurer                   Group B
                                                            10.28 Cell Culture Lab Assignment #1
3

1. Determine the cell density in the old spinner flask using the Cedex. Calculate the volume of
cells necessary from this flask to achieve the desired cell density in the new cell flask.
2. Enter the tissue culture room wearing long pants, closed-toed shoes, lab goggles or glasses
with plastic lenses, a lab coat, and lab gloves. Remove MethA medium from the refrigerator and
warm in a 37 oC water bath for 20 minutes. The medium should have an orange color, indicating
that it has a pH near 7.0.
3. Spray hands with a copious amount of 70% ethanol. Spray the medium bottle, the old spinner
flask, and a new spinner flask with 70% ethanol, then bring these into the biosafety hood.
Loosen the lids on the bottle and the flasks as much as possible. Try to keep the lids on, but not
tightened, as much as possible while working in the hood.
4. Spray a package containing a pipet with 70% ethanol and bring into the biosafety hood. Open
the package at the top and insert the pipet into a pipetter. Remove the package from the pipet. In
working with the pipet, try to avoid touching its tip to anything in the hood.
5. Transfer the calculated volume of cells from the old spinner flask to a 50-mL centrifuge tube.
Centrifuge at 1,000 rpm for 5 minutes at room temperature.
6. Using the Pasteur pipet connected to the pump beside the biosafety hood, remove the
supernatant from the centrifuge tube. Using the pipet and pipetter in the hood, add 10 mL of
fresh, pre-warmed medium to the centrifuge tube. Resuspend the cells by repeatedly sucking
them into the pipet and releasing them until there are no white patches left in the medium. Do
this gently, trying to create as few bubbles in the medium as possible.
7. Pipet the calculated volume difference of medium into the new spinner flask. Add the
suspension from the centrifuge tube.
8. Close the lid of the spinner flask tightly, but leave the caps on the sidearms open at least one-
half turn.
9. Label the spinner flask.
10. Place the spinner flask on a magnetic stirrer
11. Remove all used materials from the hood. Spray the work area with ethanol and wipe down.
Close the hood and turn on the UV light.
12. Clean the old spinner flask in the sink.
4

a.
                                             E. Coli                           CHO
average size (m)                1–5                              8 – 10

cell wall?                       yes (thin, gram-negative)        no

average doubling time (hr)       .333 - .5                        12 – 20

average cell concentration at    50                               1
the end of a process (g/L)
recombinant protein location     intracellular                    extracellular (secreted)

recombinant protein              unfolded                         folded
conformation
post-translational               No                               yes, including glycosylation
modifications?
disulfide bonds?                 No                               yes


b. The recombinant EPO produced by process A was likely translated in a CHO cell or other
mammalian cell. In mammalian cells, recombinant proteins can undergo post-translational
processing such as glycosylation. This processing will add tags to the ends of the protein that
will prevent it from being treated as waste material and flushed from the body or destroyed by
proteases in the blood. The EPO produced by process B was likely translated by an E. Coli cell
or other bacterial cell, where it could not have undergone post-translational glycosylation or
other processing. This would leave the protein vulnerable to proteases in the body and thereby
greatly reduce its half-life.
c.
                                              STR                                     Wave

mixing method                   stirring with impeller             rocking mixes in about 7
                                                                   seconds
aeration method                 sparging from bottom of            rocking increases dissolved
                                reactor                            gas levels, diffusion at surface
O2 mass transfer coefficients   10-2                               10
(1 / s)
scalability                     1 – 50000 L                        .1 – 500 L

pH control                      controller can increase dCO2       controller can turn acid (HCl) / buffer
                                                                   (NaHCO3) solution pumps on and off
                                concentration or add base
pH sensor types                 solid-state pH electrode,          specially-designed probes
                                optical fiber pH sensor            insert into luer port on bag
DO control                      single large controller for dO2,   controller increases rocking
                                dCO2, base, heating                speed to maintain dO2 level
DO sensor types                 wide range of possible probes      specially-designed miniaturized probe with
                                                                   Clark-type polarographic electrodes fits into
                                                                   special sheath on bag
sterilization method            wash with ethanol, autoclave       wash with ethanol, replace bag
                                after use                          after use
for microbial cell work?        yes                                no (not enough dissolved
                                                                   oxygen)
for cell culture work?          yes (must reduce shear stress      yes
                                with additive or adherent)

				
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