Antibiotic neutralizers in sterility testing

Bob Friedel rfriedel at perrittlab.com
Thu Jun 1 11:58:07 EST 2000


Neomycin has a low concentration exponent, i.e., it will NOT undergo rapid
neutralization by dilution.  Neutralizing agents (e.g., Tween 20/80 and
Lecithin) used in the USP Microbial Limit Tests are normally used to
inactivate preservatives (e.g., parabens et al.) and the level of
effectiveness with antibiotics is limited/absent.  Antibiotics are generally
inactivated ENZYMATICALLY.

Check the following references:

Russell, A.D., and Rogers, D.T. (1984) "Laboratory Uses of
Antibiotic-Inactivating Enzymes", J. Antimicrob. Chemother., Vol. 14, p.
567.

Corry, J.E.L., Van Doorne, H., Mossel, D.A.A. (1977) "Recovery And Revival
Of Microbial Cells, Especially Those From Environments Containing
Antibiotics", In: Antibiotics in Agriculture, M. Woodbine (ed), Chapter 12,
pp.177

Negretti, F. (1989) "Experimental Observations on the Bacteriological
Controls of the Antibiotics--I. Antibacterial Activity of Membranes Employed
in Bacteriological Assays", J. Pharma. & Biomed. Anal.,  Vol. 7, No. 12, p.
1861.

Negretti, F. and P. Casetta (1991) "Research on Sterility and Contamination
Controls of Chemotherapeutic agents by Membrane Filtration Method", J.
Pharma. & Biomed. Anal., Vol. 9, No. 9, p. 773.

Options include the following and may be used in combination:

a) adding an antibiotic neutralizing/inactivating agent to the broth/plating
media

b) increasing the concentration of the antibiotic neutralizer/inactivator in
the broth/plating media

c) physical separation of the organisms from the antibiotic (i.e., membrane
filtration)

Use an alternate membrane filter media (e.g., polyvinylidene difluoride
[PVDF] or polyethersulfone [PES]), as cellulose-based membranes tend to bind
antibiotics.  If the organism(s) do not grow on the antibiotic-filtered
membrane, there is still activity within the membrane (assuming that the
membrane material itself was checked for antimicrobial activity in the
absence of the antibiotic).  In addition, if the antibiotic is still bound
to the CN or CA membrane, it may be released upon introduction to a broth
medium.  The rest is self explanatory.  This may be eliminated by changing
the filter material (e.g., PVDF or PES) and/or adding chemical neutralizers
to the rinse medium.  As part of your validation protocol, you should add a
low number of organisms prior to the final rinse (obviously a control is
included).  The antibiotic inactivator should have contacted the filter for
a predetermined time prior to addition of the inoculum.

Two new standards for membrane filtration counts have been developed (ISO
Standard Development Group TC172/SC7/WG5).  The count ranges for bacteria
and yeast are 3-100 cfu/filter and 3-10 cfu/filter for mold (47 mm diameter
filter membrane).  Keep in mind that plate counts are notoriously inaccurate
particularly at low levels and should be viewed only as estimates.

d) increase the volume of the rinse

At some point in the process, you may need to state that the material under
consideration has enough intrinsic antimicrobial activity to warrant the
exclusion of the Microbial Limit Tests.

Your best option is a combination of the membrane filtration method with
chemical neutralization.

Bob Friedel
Quality Assurance Manager
Perritt Laboratories
Hightstown, NJ 08520 USA
(609) 443-4848
http://www.perritt.com
rfriedel at perrittlab.com

Austin Reade <rbs at hfx.andara.com> wrote in message
news:I_9Z4.47$WA.8887 at sapphire.mtt.net...
> A client has asked for my advice on sterility testing a pharmaceutical
> product.  As this is out of my field I wonder if anyone on this newsgroup
> has any suggestions.
>
> The product contains neomycin sulphate and chloramphenicol.  They find
this
> inhibits growth of their positive control cultures when even the smallest
> (reasonable) quantity of product is added to thioglycollate broth.  They
> want to know if there are any specific neutralizers for these antibiotics.
> I suggested the possibility of using membrane filtration but they are
> reluctant to use mf because of the additional labour involved.  I also
> recall some previous postings here on the problems of binding of some
> antibiotics to certain filter types.
>
> Any thoughts would be appreciated.
>
> Austin.
> --
> Austin Reade, PhD, RSM(CCM)
> Reade BioSciences Inc                 phone (902)423-8369
> 1136 Cartaret Street                       fax (902)423-8313
> Halifax, NS, B3H 3P3 Canada      email rbs at hfx.andara.com
>
>
>







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