Drugs and the pharmaceutical sciences

Prefiltration in Biopharmaceutical Processes 17
FIGURE 16 Plasma fractionation.
separated by centrifugation. It contains the antihemophilic factor, fibronectin (coldinsoluble
globulins), and fibrinogen.
The cryo-poor plasma pool is then adjusted with regard to pH, and cold ethanol is
added to a specific concentration. The pH is usually 7.4 ± 0.2, the alcohol concentration
8%, and the temperature –2 ± 0.5˚C. This treatment yields a fraction I precipitate, and a
supernatant I.
Treatment of supernatant I with 20% ethanol at pH 6.8–6.9 at –5 ± 1.0˚C yields
fractions II and III precipitate plus supernatants II and III. Adjustment of the pH of
supernatant to 5.2 ± 0.1 at –±1˚C results in fraction IV precipitate. If treated at pH
5.9 ± 0.05 at the same temperature with ethanol at 40%, the fraction IV4 followed by pH
adjustment to 4.6–4.8 at –± 0˚C and the addition of ethyl alcohol at 40% yields fraction V
as a precipitate along with the supernatant V.
Another common procedure for obtaining the plasma protein fractions is to
combine fractions I, II, and III at 20% ethanol, fractions IV1 and IV4 at 40% alcohol, and
fraction V at 40% alcohol. The point being made is that there are variations of the original
Cohn fractionation that are practiced.
Albumin
In essence, fraction V is crude human albumin. It contains salts and residual alcohol. It
is dissolved in distilled water, and made up to 10% ethanol concentration at pH 4.6–4.8 at
3 ± 1˚C. It is then passed through a depth filter to remove any extraneous, insoluble
proteins. The filtered solution is readjusted with alcohol to 40% strength at pH 5.2 þ 0.1
at 6 ± 1˚C. A supernatant is removed by centrifugation to leave a precipitate of albumin.
The reworked albumin precipitate is dissolved in distilled water, prefiltered through
3–8 mm-rated depth filters, and final filtered through 0.2-mm-rated membranes.
The albumin solution is then concentrated, usually by one of three methods, namely,
lyophilization followed by reconstitution with sterile water, by thin-film evaporation, or
by ultrafiltration. Where lyophilization and reconstitution are utilized, the resulting
nonsterile bulk solution is stabilized and again filtratively sterilized by passage through
depth and final filters into a final sterile bulk tank. Where thin-film evaporation is used,
the resulting fraction V precipitate is dissolved in distilled water, stabilized, and clarified
by use of prefilters before being subjected to the thin-film evaporator. Following this, the
concentrated solution is again prefiltered and final filtered through a sterilizing
membrane. Where ultrafiltration is employed as the means of concentration, the
procedure is the same except that ultrafiltration is substituted for the thin-film evaporator.