|
Cleaning-In-Place (CIP) and Validation.
Unlike competition, SPC
columns assure fast and complete CIP
as they do not have stagnant
pockets blocking the flow through the
frit. -
Our customers report 3-4-fold faster equilibration time and reduced
buffer consumption (see References). The
columns are made for Biseps by manufacturers catering to bio- and pharmaceutical
industry and meeting 3A, FDA, ISO-9001 and USDA requirements.
-
CIP
protocols and validation results can be confidently translated from a
small to a large SPC since the stagnant distance is kept low - throughout all column sizes.
Useful facts:
Stagnancy can lurk in the following places:
 1. Under the mesh frame and/or next to the O-ring. Depending on the size of the
conventional column, this "black nail rim" can be 3 to 35-mm
deep and is responsible
for up to 100--X variation
in equilibration time during the scale up.
In
contrast, SPC columns have open mesh extending all the way to the wall.
About 2 to 5-mm of the mesh edge is tucked under the gasket which results
in more consistent CIP throughout the scale up.
2. Dead space under the packing
valve (over 50-mm diameter). In contrast to the
"valve-through-the-lid" design, SPC packing port closes flush with the wall
so that the flow through the entire mesh surface is unobstructed.
3. Screws: even a small 1/4"-28 screw 1/4"-long has a spiral channel about 5"-long!
In
contrast, SPC design stays away from the dead-ended coils.
|
| Theoretically,
the time required for a certain
concentration change is proportional to
the length of the stagnant pocket in the power of
two. |
| 3-log concentration change (CIP examples) |
3-mm (1/8") |
6-mm (1/4") |
25-mm (1") |
| NaOH |
3-min |
12-min |
3.2-hrs |
| Binding biopolymer |
2-hrs |
8-hrs |
5-days |
|
Long stagnant pockets slowly
ooze their contents into the mainstream. The contamination is hard to detect as
it gets diluted millions of times. Stagnant
spaces can manifest themselves as:
a) Bacteria and microbe release endotoxins, proteases and
other contamination, and/or degrade the product;
b) A "strange" pH or UV bump appears after a
"perfectly equilibrated" column was allowed to stand for a while ( e.g.
when the column was soaked with 2 M NaOH, then equilibrated with a weak buffer
and stood overnight).
|
 Fig.1.
Making covert - visible. Simulated stagnant pocket
(0.01% CV by volume and 0.5-cm deep) was placed next to the transparent wall
of a 36-cm ID column. One can
clearly see
"bleeding" of the leftover load (colored RBC extract). However, even
a sensitive enzymatic assay fails to detect this contamination in the washing
buffer due to
about 7-log dilution of the protein (courtesy of Lee
Scientific, Inc., see References).
More data on the "invisible" contamination is
presented on Fig. #2 on the "Data
& Graph" page.
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|
Column surfaces. |
| For example, let us consider a 20-L stainless steel
column, 36-cm ID, 20-cm height, equipped with 35-um frits. |
SURFACE AREA
|
| Lid (2) |
2036 cm2 |
| Wall |
2262 cm2 |
Mesh (2)
(e.g. 400x400 mesh, with a 0.0010" wire) |
6434 cm2
|
Conclusions:
1. Mesh comprises about 2/3 of the column surface and cannot be
"electro-polished".
2. Therefore, there is little sense for electro-polishing the lids and the
wall only.
3. Column re-use: replace the mesh as opposed to "flossing out"
the old sorbent. |
|
Biseps,
Inc. Advanced
Columns and Methods for Preparative Bio-Chromatography
