Use of sodium hydroxide for cleaning and sanitizing


Sodium hydroxide is widely accepted for cleaning,
sanitizing, and storing chromatography media and
systems. The benefits of its use include efficacy, low cost,
ease of detection, removal, and disposal. As with any
sanitizing agent, certain precautions should be taken
and compatibility with both chromatography media and
systems determined.
This Application Note examines these aspects of using
sodium hydroxide as a cleaning and sanitizing agent. It
includes many examples of particular interest to producers
of recombinant proteins, monoclonal antibodies, and
oligonucleotides, since designing and scaling up validatable
cleaning processes is a critical issue in the commercial
manufacture of these products (1).
Efficacy
Sodium hydroxide has been shown to be effective in
removing proteins and nucleic acids. It is also effective
for inactivating most viruses, bacteria, yeasts, fungi,
and endotoxins. It is common practice in industrial
manufacturing to save time by adding a salt, such as
sodium chloride, to the sodium hydroxide solution to
combine cleaning with sanitization.
Removal of proteins and nucleic acids
As a cleaning agent, sodium hydroxide saponifies fats and
dissolves proteins (2). In general, it can solubilize precipitated
proteins. Its hydrolyzing power is enhanced by the presence
of chlorine (3).
The ability of sodium hydroxide to remove proteins and
nucleic acids from chromatography media depends on the
nature of the media, the sample, and sample contaminants
that may interfere with the cleaning efficiency. For example

a higher concentration of sodium hydroxide may be
required if lipids are bound to a protein. To demonstrate
the effectiveness of the sodium hydroxide solutions, users
should periodically sample the stored solutions and also run
blank gradients after cleaning (4).
Proteins
Sodium hydroxide has been used extensively to remove
proteins from ion exchange, hydrophobic interaction, and
gel filtration media. Traditionally, the use of sodium
hydroxide with affinity chromatography media has been
restricted due to the limited stability of most immobilized
ligands. Today, recent developments have changed that
picture and modern Protein A-based affinity media, such as
MabSelect SuRe™ for monoclonal antibody purification, have
significantly improved compatibility with sodium hydroxide.
MabSelect SuRe, with its alkali-stabilized protein A-derived
ligand, was designed to withstand 0.1 to 0.5 M sodium
hydroxide solutions as cleaning agents.
Nucleic acids
Nucleic acids can bind tenaciously to anion exchangers.
However, work in our laboratories has shown that a
combination of 1 M sodium hydroxide and 3 M sodium
chloride, with a total contact time of one hour, effectively
removes radiolabelled calf thymus DNA from DEAE
Sepharose™ Fast Flow, a weak anion exchanger. A small
percentage of the radiolabelled DNA was retained, and
could not be eluted under any conditions tested. Others
have found that lower concentrations or shorter contact
times are insufficient to remove nucleic acid from DEAE
Sepharose Fast Flow and restore the medium’s separation
capabilities.
Further work on Q Sepharose Fast Flow, a strong anion
exchanger, has shown that 1 M sodium hydroxide combined
with 1 M sodium chloride effectively removes DNA, but that
the level of removal is dependent on the nature of the sample
(5). The contact time for cleaning-in-place (CIP) in these
experiments was 2 hours. For one sample, DNase was required
to completely remove DNA from the anion exchanger.

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