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68 Gallant et al.
2. Adjustment of the load proceeds in two steps: pH and conductivity adjustment,
followed by clarification. The pH should be adjusted using a dilute acid (10%
acetic acid) or base solution (1 M Tris base), depending on whether the pH is to
be decreased or increased. Conductivity should be adjusted by adding a concentrated
sodium chloride solution (4 M) or deionized water, depending on whether
conductivity is to be increased or decreased. Clarification to a final 0.2-μm filtration
can be accomplished at small scale by centrifugation followed by vacuum filtration
with a glass fiber prefilter (provided that the protein of interest does not bind to
the glass prefilter). A glass fiber free filter train which works quite well for most
mammalian cell culture supernatants consists of the Sartorius Sartopure PP2 1.2-μm
filter followed by a Sartopore 2 0.45/0.2-μm filter. Some thought should be given
to the filtration method because product losses can be quite large if an inefficient
method of filtration is selected.
3. In some cases, a chromatographic system may be unavailable or undesirable. The
later case occurs with a feedstock which may be inappropriate to contact with a
system which is used repeatedly (e.g., a load sample containing virus). In that case,
a peristaltic pump with disposable tubing is used to pump the solutions. Small
discrete steps in buffer concentration can be substituted for a linear gradient. Three
column volumes of 10% B, then 3 column volumes of 20% B and so on. Column
fractions are analyzed by UV spectrophotometer.
4. Selection of the appropriate amount of resin per batch binding experiment is
important to the success of the experiment:
a. A typical expression level of a recombinant protein in mammalian cell culture is
0.02 mg/ml of supernatant (although some titers may be 10-fold above or below
this). For low titer cell culture fluid, a concentration step (ultrafiltration) may
be desirable in order to reduce the volumes of feedstock needed in the batch
binding experiments.
b. A desirable binding capacity for capture of a protein from cell culture is 4 mg/ml
of gel (although 5-fold above this is possible for high affinity proteins).
c. In order to load 50 ml of resin to 4 mg/ml with a 0.02 mg/ml cell culture
supernatant, 10 ml of cell culture supernatant will be required for each condition
to be tested.
Loading of the gel in batch binding experiments should be substantial or it will be
difficult to interpret the results. Overloading the resin is not generally a problem.
Conversely, underloading the resin provides only limited data regarding the binding
capacity for the target protein and may lead to poor recovery (accountability).
5. If necessary, dialysis at 5ºC may be used to preserve protein activity. Disposable
desalting columns may be used to reduce processing time. If precipitation is
observed during dialysis, do not terminate that experimental condition. After dialysis
is complete, clear the precipitate by centrifugation and continue with the binding
experiment using the clarified supernatant. Frequently, the protein of interest
remains in solution; a specific assay (activity in the case of an enzyme) is required
to verify loss of the protein of interest.