Improving microbiological assurance for bioburden testing

Reference:

Sandle, T. (2016) Improving microbiological assurance for bioburden tests, European

Pharmaceutical Review, 21 (3): 41-44

Introduction

Assessment of microbial levels in (and on) samples is an important part of pharmaceutical

process control. Samples are drawn from intermediate product at defined stages (ideally
based on risk assessment) and these allow for the microbial levels to be tracked from
upstream processing to downstream processing (with an expectation that the microbial levels
decrease, or at least remain unchanged provided they are below an acceptable action level).
For aseptically products, European guidelines require a certain bioburden to be met at the
point that a bulk product passes through a sterilising grade filter.

Due to the relatively low specification – of 10 CFU/100mL – pharmaceutical manufacturers

need to ensure that false positive results are avoided (as might arise from extraneous
environmental contamination). False positives can result in batch rejection. A key innovation,
in recent years, is the biocontainer sampling bag. This item of irradiated plastic is in keeping
with moves towards single use, sterile processing technology.

This article examines the importance of bioburden testing, particularly in relation to

aseptically filled products, together with the most important criteria for sampling bags.

Bioburden

Bioburden refers to the microbial content of a material (or on the surface) at a given point in
time. This could be prior to sterilisation or in relation to a process hold time (1). Bioburden
refers to an estimation of the numbers of bacteria and fungi present in a liquid sample.

Bioburden is an important activity in relation to the assessment of microbiological control.

With pharmaceutical processing, bioburden is undertaken to assess the microbial levels in
intermediate product samples. This allows an assessment to determine:

If levels of bioburden are higher at the start of the process (upstream samples)
compared with later in the process (downstream samples).
If parts of the process are considered to lead to bioburden reduction are effective.
Whether additional process steps, such as water rinses, contribute to the bioburden.
Where applicable, if additives to the process, such as formulation buffers, contribute
to the bioburden and these are not filtered.

All of the intermediate products should be considered non-sterile. Given the nature of the
material, a level of microbial recovery is expected. However, regulatory agencies expect
good bioburden control throughout the manufacturing process (2).

In addition, the collected bioburden levels are of interest in terms of long-term trending for:

a) Proportion of samples that exceed an alert or action level;

b) Changes in the total or mean count;
c) Changing profile in relation to the types of microorganisms recovered.

In order to improve the meaningfulness of the sample, samples should be taken at the end of
any hold period since this will represent ‘worst case.’ It is also important that the sample
taken is representative of the process stage, in terms of homogeneity.

Bioburden testing is commonly performed using the Total Aerobic Microbial Count method,
either using a pour plate agar method or, more preferably, membrane filtration. Rapid and
alternative microbiological methods are available. With the agar methods, care needs to be
taken in relation to selecting an appropriate agar (commonly tryptone soy agar) and with
incubation time and temperature, in order to ensure optimal recovery (3). Recovery
assessments are performed using low-level microbial challenges. Collected samples will need
to be assigned an expiry time (which will need qualifying) and storage would ordinarily be at
2-8oC in order to slowdown or suspend the rate of microbial growth.

In relation to aseptic processing, the bulk product requires bioburden determination prior to
final filtration. The material, at the point of final sterilising grade filtration, must be of a
sufficiently low bioburden as not to present an unduly high challenge to the sterilising grade
filter. This activity is considered below.

Pre-final filtration bioburden

The most important sample in relation to aseptically filled products is the sample taken of the
bulk product prior to final filtration. Final filtration involves passing the bulk through a 0.2
µm sterilising grade filter, into a sterile vessel. After mixing, the sterile homogenous bulk is
aseptically filled. With filtration, the Parenteral Drug Association guidance document
"Sterilizing Filtration of Liquids" published as Technical Report No. 26, defines sterilising
filtration as "the process of removing all micro-organisms, excluding viruses, from a fluid
stream".

Although there is no comparable limit in the U.S., within Europe a maximal limit is in place
for aseptically filled products at the point before the bulk passes through the sterilising filter
(4). With this, a CPMP Note for Guidance on Manufacture of the Finished Dosage Form has
a section relating to the appropriate bioburden level of the bulk solution prior to sterilisation
by filtration, for products distributed in the European Union. This reads (5):

“For sterilisation by filtration the maximum acceptable bioburden prior to the filtration must
be stated in the application. In most situations NMT 10 CFU’s/100 ml will be acceptable,
depending on the volume to be filtered in relation to the diameter of the filter.”

The U.S. FDA guidelines on aseptic filling state that a suitable limit must be set (6):

“Manufacturing process controls should be designed to minimize the bioburden in the

unfiltered product. In addition to increasing the challenge to the sterilizing filter, bioburden
can contribute impurities (e.g., endotoxin) to, and lead to degradation of, the drug product. A
prefiltration bioburden limit should be established.” However, no actual value is specified.

The origin of the CPMP limit appears to have a relationship with the recommended microbial
count for bulk WFI (not more than 10 CFU / 100 ml); and with consideration of the standard
sterilising filter rating of the retention of 107 colony forming units of the challenge micro-
organism Brevundimonas diminuta per cm2 of the filter surface (based on a 0.2µm filter pore
size). The reason why Brevundimonas diminuta is selected for filter challenges is (7):

Originally a process stream isolate, and therefore representative of potential

contaminating organisms in parenteral manufacture.
Generally regarded as non-pathogenic to humans, thus minimising safety concerns for
personnel involved conducting challenge tests.
The micro-organism can be consistently cultured under controlled conditions to
produce very small, monodispersed cells with a narrow size distribution which can
penetrate 0.45μm filters reproducibly in small numbers at high challenge levels.
Under these conditions the organisms, thus represents a potential worst-case
challenge.

The use of 10 CFU / 100 ml provides a theoretical limit 180 – 800 times lower than the
standard sterilising filter rating and 3 – 4 magnitudes lower than the 104 – 105 Gram-negative
bacteria / mL that theoretically present enough bacterial endotoxin to elucidate a pyrogenic
response (8).

Many product licences require strict adherence to the bioburden limit. If this is exceeded,
irrespective of any reference to filter validation bacterial challenges, the product may be
rejected. For this reason, care is taken with sampling the bulk for laboratory testing. Simply
eluting the product into a sterile universal container may not be sufficient, no matter how well
someone has been trained, for occasional adventitious contamination can occur. Such
contamination can be difficult to prove and a decision often needs to err in favour of the
result being indicative of the bioburden load of the product, leading to batch rejection.

Single-use sterile technology

This decade has seen a number of innovations with single-use sterile technology.
Applications include tubing, capsule filters, single-use ion exchange membrane
chromatography devices, single-use mixers, and bioreactors, product holding sterile bags in
place of stainless steel vessels (sterile fluid containment bags), connection devices and
sampling receptacles (9). Reasons for research and innovation into these types of technology
includes: reducing processing time (which is linked to economic pressure to improve time-to-
market); to reduce manufacturing costs, to go for manufacturing systems which are more
reliable, flexible, cost effective and to seek improved sterility assurance. Biocontainer bags
form part of the sterility assurance application (10).

Biocontainer sampling bags

The conventional way to sample the bulk product for bioburden is to withdraw a quantity of
the material from a holding vessel, using a valve or syringe, and to transfer this to a sterile
sampling container. This process, which is operator dependent and involves multiple steps,
poses a risk of adventitious contamination and thus of a false positive result being reported
which, at a sufficiently high bioburden, could lead to product rejection. Due to the integral
nature, the use of single-use, sterile biocontainer bags allows the sample to be taken in a way
which eliminates the possibility of external or operator contamination triggering a false
positive result.
With biocontainer bags there are a number of considerations. These are:

a) The bags should be sterile, using a process that does not damage the plastic material.
This is normally by gamma radiation.
b) The integrity of the bags should not deteriorate over time (discoloration or loss of
material strength).
c) The plastic material should not react with the sample. The optimal condition is that
the plastic is chemically inert. Here the bags should be assessed for any leachables or
extractables which might arise when the product comes into contact with the single-
use technology. The presence of extractables, in particular, could lead to adulterated
product or to the inhibition of any microbial contamination (leading to a false
negative result) (11). Inhibition of microbial growth is of great concern. This is
because any inhibited microorganisms, within the product, could move from a state of
dormancy to one of exponential growth once the product is no longer in contact with
the single-use item.

An assessment of inhibition can be made by running a microbial challenge study

whereby product held in the bag is challenged with a low level (<100 CFU) of a
microorganism. The challenged product is held for a period of time which
approximates the filtration time, after which samples are taken and the level of
microorganisms assessed. The absence of inhibition is confirmed by the suitable
recovery of the challenge microorganism at a level close to the initial challenge (such
as between 50 and 200%). This study should be repeated for different types of
microorganisms which are representative of the typical cleanroom environmental
flora (such as Gram-positive coccus like a Staphylococcus spp., a Gram-positive rod
such as a Bacillus spp., a Gram-negative rod such as a Pseudomonas spp., and a
fungus, such as an Aspergillus spp.).

d) The bag should be suitable for the product, as assessed against time and temperature.
Physicochemical testing should capture the storage times of the product and include
the storage conditions (such as holding temperatures).
e) The bag should be graduated, so that the correct volume of fluid is taken each time.
f) The use of the bag, for sample collection and sample testing, should be easy for
operators.
g) The bags should be larger than the volume of product required for the sample, so that
sufficient air space remains (which helps with the removal of the sample),
h) The bags should be equipped with aseptic connectors. It is important that the bags
have aseptic connectors, and a means for the sample to be removed aseptically once it
is back in the testing laboratory. An aseptic connection allows fluid to be passed from
one vessel to another in a way which does not introduce microbial contamination.
Innovations in aseptic connection technology have led to the development of single
use connector systems to allow for a totally enclosed and automated process.

The above factors can be used to develop a supplier technical document or form part of an
audit checklist.

Summary

This article has explained the importance of bioburden testing in terms of microbiological
process control. The avoidance of false positives in relation to this important test can be
avoided through the use of biocontainer sampling bags, which as a side-application of single-
use sterile technology. The qualification of the bags, to show that they do not possess any
inhibitory properties, together with a reliable method for collecting and removing the sample,
are necessary. These must form part of good design practice. Once these issues have been
assessed and demonstrated, biocontainer bags present a useful way of enhancing
microbiological assurance in pharmaceutical manufacturing and can reduce the number of
false positive counts.

References

1. Sandle, T. (2015) Assessing Process Hold Times for Microbial Risks: Bioburden and
Endotoxin, Journal of GXP Compliance, 19 (3): 1-9
2. Sandle, T. (2012). Review of FDA warning letters for microbial bioburden issues
(2001-2011), Pharma Times, 44 (12): 29-30
3. Sandle, T., Skinner, K. and Yeandle, E. (2013). Optimal conditions for the recovery
of bioburden from pharmaceutical processes: a case study, European Journal of
Parenteral and Pharmaceutical Sciences, 18 (3): 84-91
4. Yang H, Li N, Chang S. (2013) A Risk-based Approach to Setting Sterile Filtration
Bioburden Limits, PDA J Pharm Sci Technol.;67(6):601-9
5. The European Agency for the Evaluation of Medicinal Products Committee for
Proprietary Medicinal Products (CPMP) Note for Guidance on Manufacturing of the
Finished Dosage Form, CPMP/QWP/486/95
6. FDA. Guidance for Industry Sterile Drug Products Produced by Aseptic Processing
—Current Good Manufacturing Practice, U.S. Food and Drug Administration,
Bethesda, MD, USA
7. Jornitz, M.W., Akers, J.E., Agalloco, J.P., Madsen, R.E. and Meltzer, T.H. (2003):
‘Considerations in Sterile Filtration. Part II: The sterilising filter and its organism
challenge: a critique of regulatory standards’, PDA Journal of Pharmaceutical
Science and Technology, 57 (2): 88-95
8. Cundell, A. M. (2004): Microbial Testing in Support of Aseptic Processing,
Pharmaceutical Technology, June 2004, pp56-64
9. Sandle, T. and Saghee, M.R. (2012). Application of Sterilization by Gamma
Radiation for Single-Use Disposable Technologies in the Biopharmaceutical Sector,
Pharmaceutical Technology, 36 (5): s20-s30
10. Sandle, T. (2013). Single-use technology for biopharma, Cleanroom Technology, 21
(12): 15-19
11. Samavedam, R.; Goldstein, A.; Schieche, D. (2006). Implementation of disposables:
Validation and other considerations. Am. Pharm. Rev., 9: 46 –51