Sandle, T. (2015) Assessing Process Hold Times for Microbial Risks: Bioburden and
Endotoxin, Journal of GXP Compliance, Vol. 19, Issue 3, Oct 2015, pp1-9
Assessing process hold times for microbiological risks: bioburden and endotoxin
By Tim Sandle
Introduction
During biopharmaceutical manufacturing various stages in the manufacturing process require
hold stages. This can be for product formulation reasons, equipment issues, or relating to
personnel change-overs. By far the most common reason will be for processing. When
processes are held there are a number of potential risks, relating to chemical stability and
from microbiological growth. The focus of this article is on microbial risks.
Microbial risks include bioburden and bacterial endotoxin. Bioburden assessment informs the
manufacturer about both the expected microbial load of the product and the presence or
absence of specific microorganisms, some of which might be classed as ‘objectionables’.
Endotoxin informs about a risk involving a specific type of microorganism (Gram-negative
bacteria) and the presence of a pyrogenic toxin that will be difficult to eliminate, if possible at
all.
Due to the potential for microbial growth, pharmaceutical manufacturers typically conduct
studies to define acceptable hold times for process intermediates. These studies are based on
the microbiological examination of in-process production samples (1). This article assesses
why hold time studies are important, in relation to the risks from microbial contamination,
and assesses those factors that lead to microbial growth. The article also discusses the
importance of fitting hold time into process validation and how hold times need to be
accounted for within a biocontamination strategy. To begin the overview, the article first
considers the regulatory requirements.
Regulations
The importance of hold time assessment, and the associated microbiological risks, is referred
to in several Good Manufacturing Practice (GMP) guidelines and in compendia. The most
important of these are:
a) Code of Federal Regulations
The key CFRs are:
21 C.F.R. 211.110 (a)(6), which states that bioburden in-process testing must be
conducted pursuant to written procedures during the manufacturing process of drug
products.
21 CFR 211.46(b) states that “Equipment for adequate control over air pressure,
micro-organisms, dust, humidity, and temperature shall be provided when appropriate
for the manufacture, processing, packing, or holding of a drug product.”
21 CFR 211.63 states that “Equipment used in the manufacture, processing, packing,
or holding of a drug product shall be of appropriate design, adequate size, and suitably
located to facilitate operations for its intended use and for its cleaning and
maintenance.”
21 CFR 211.160(b) – laboratory controls - discusses the "determination of
conformance to appropriate written specifications for the acceptance of each lot
within each shipment of components, drug product containers, closures, and labeling
used in the manufacture, processing, packing, or holding of drug products."
21 CFR 211.111 states that “When appropriate, time limits for the completion of each
phase of production shall be established to assure the quality of the drug product.
Deviation from established time limits may be acceptable if such deviation does not
compromise the quality of the drug product. Such deviation shall be justified and
documented.”
b) U.S. Food and Drug Administration (FDA)
Examples of the FDA emphasis on hold times are found in the FDA guidance on aseptic
processing (2). For example, the guidance highlights:
In relation to processing, it is a requirement that sterilized holding tanks and any
contained liquids should be held under positive pressure or appropriately sealed to
prevent microbial contamination.
Written procedures should specify the frequency of revalidation of processes as well
as time limits for holding.
Importantly the guidance requires that "drug product components, containers,
closures, storage time limitations, and manufacturing equipment are among the areas
to address in establishing endotoxin control."
Validation is referred to when the guidance notes: "the time limits established for the
various production phases should be supported by data. Bioburden and endotoxin load
should be assessed when establishing time limits for stages such as the formulation
processing stage."
Furthermore, lack of bioburden control or bioburden action level excursions are regularly
cited during FDA inspections, as review of warning letters indicate (3). A common citation is
for a lack of data to support process holding times in relation to microbial contamination (4).
c) United States Pharmacopeia
In the USP <1115> chapter "Bioburden control of nonsterile drug substances and products"
(5), the recommendation made is that “manufacturers should consider whether processing
steps and hold periods could result in changes to bioburden.” Furthermore, manufacturers
must “properly establish processing hold times.”
d) EU GMP
European Good Manufacturing Practice, in Annex 1 which pertains to sterile product
manufacture (6), suggests that "the time between the start of the preparation of a solution and
its sterilisation or filtration through a micro-organism-retaining filter should be minimised."
This sentence emphasises the risk of microbial growth at key stages. The guide goes onto to
state: "there should be a set maximum permissible time for each product that takes into
account its composition and the prescribed method of storage."
The selections from GMP guidances indicate the risks that can arise at different process
stages and how these risks can increase when the process is held. Why might microbial risks
arise?
Microbiological risks
Pharmaceutical preparations, especially biologic products, are at risk from microbial
contamination many stages. Such risks exist because biopharmaceuticals often include the
types of carbon sources and other growth factors that favour microbial growth. Moreover,
many of the types of microorganisms found within the environment, including process areas
can adapt and survive under a variety of conditions. Where microorganisms are capable of
growth in conditions that favour cellular division, then microbial contamination poses a
significant risk to biologic products.
Microbial risks arise from a variety of sources. These include the facility, equipment, process
operations, raw materials, column resins, filter membranes, water, process gases, and
personnel (7). These present areas where a breakdown in control can lead to microorganisms
being present. Further weaknesses in control measures can lead to ingress of microorganisms
into the product or formulated excipients. With process there are many variables where
contamination can occur. For example, open processing presents a greater contamination risk
than closed processing. Here open processing may be an individual event, or it could be that a
vessel is opened several times for mixing or the addition of chemicals. The room
environment and operator aseptic practices will also impact upon any assessment.
Whether microorganisms survive or proliferate is based on several physicochemical factors.
Thus the outcome, following microbial ingress, is either survival without growth, growth, or
death. These outcomes are dependent upon product, process, time and temperature. With
products and processes, some products and intermediates will be at more of a risk than others.
For example, biopharmaceuticals or therapeutic protein products are derived from
recombinant DNA and hybridoma technology; such materials are at a greater risk than
inorganic additives. Chemicals added to the direct product, raw materials, media, buffer
solutions, in-process intermediates and are also generally growth promoting.
Microorganisms need substances for energy generation and cellular biosynthesis. These are
obtained from different growth sources (8). Many bacteria utilise carbon. Such organisms are
divided into heterotrophs, which use organic molecules such as sugars, amino acids, fatty
acids, organic acids, aromatic compounds, nitrogen bases, and other organic molecules for
their source of carbon; and autotrophs which use inorganic molecules of carbon dioxide as
their source of carbon. Other bacteria utilise nitrogen, either solely or in addition to carbon.
Other common nutritional requirements which bacteria need to utilise for growth include
phosphorus, sulphur, potassium, magnesium, calcium, sodium, and iron. The actual
nutritional types, quantities and combinations will depend on the bacterial species.
With time, bacterial growth individual cells divide in a process described as binary fission.
Here two daughter cells arise from a single cell. The daughter cells are identical except for
the occasional mutation. Exponential growth is a function of binary fission; this is because at
each division there are two new cells. The time between divisions is called generation time
(this is the time for the population to double.) Generation times can range from minutes to
several days depending on the species of bacteria. One of the fastest dividing bacteria is
Escherichia coli, which can double every 15-20 minutes under ideal conditions (9). Leading
up to the beginning of growth is a lag phase; the time that this lasts for varies depending upon
the physiological state of the organism and the conditions with which it finds itself.
Bacterial growth does not go on indefinitely; there are factors that limit population growth.
These factors include are intraspecific competition for nutrients, which reduce as the culture
ages; and the build up of toxic metabolites. When these conditions occur a stationary phase is
reached, when no growth occurs; should the depletion of nutrients or build-up of toxicity
continue, this is followed by cell death.
Certain process factors can affect growth, should contamination occur. Head-space ratio, for
example, can be important. Increased head space combined with agitation in a hold vessel can
increase product oxidation throughout the hold period. This may favour bacteria that prefer
aerobic conditions.
With temperature, there are some conditions that are more favourable to microbial growth
than others. The temperature will depend upon the type of bacteria. Bacteria that grow
optimally under different conditions are commonly divided into:
Psychrophiles - low temperature optima <15oC.
Mesophiles - midrange temperature optima 25 - 40oC.
Thermophiles - high temperature optima 40 - 80oC.
Hyperthermophiles - very high temperature optima >80oC.
Given that cleanroom contamination originates primarily from people, then mesophilic
bacteria (and fungi) are most likely and therefore they pose the biggest risk (10). This means
that processes occurring at 'room temperature' are at a greater theoretical risk.
Other factors influencing the likelihood of microbial growth include pH. With acidity and
alkalinity factors, most microorganisms prefer to grow between pH 5 and 9, with an optima at
a neutral pH of 7. A further factor is oxygen, with many aerobic bacteria (which will be the
most common) preferring aerobic conditions. To add to this, there is water activity, where
increasing dryness means fewer species of bacteria can grow or survive.
Product, process, time and temperature should not be viewed as discrete factors. These
factors often need to be combined since one factor in conjunction with another may lead to a
different risk outcome. For example, one type of growth promoting product held at 2-8oC
would be at a lower risk, due to this temperature inhibiting the growth of most
microorganisms, that the same product held for the same time period at 30-35oC.
Biocontamination control strategy
Assessment of hold times should form part of the biocontamination control strategy. Such a
strategy will centred on controlling the source of microorganisms and ensuring that
conditions that promote microorganism survival, growth and persistence are minimized. In
the context of hold times this will be through reducing the hold time, if possible, and ensuring
that established hold times are qualified. The strategy should also assess whether measures
are in place to minimise the possibility for the survival or growth of microorganisms (11).
This is achieved through control of in-coming materials (and testing); storage; preparation of
solutions; assessment of water quality; equipment cleaning; and personnel controls, including
gowning and activities (12).
Aspects to consider as part of the biocontamination control strategy include (13):
Environmental controls for the cleanrooms in which processing takes place, including
the grade of the area.
Mapping equipment and personnel flows.
Process conditions, especially those to minimise microbial growth, such as
temperature.
Frequency of environmental monitoring in cleanrooms where the product is
processed.
Environmental monitoring methods, and locations for monitoring.
Consideration of the types of process samples to take (from the intermediate product
and from buffers and excipients); the process times when the samples need to be
taken; and the types of tests required (bioburden and / or endotoxin.)
Once all controls are in place, the strategy will need to determine a test regime: which
samples should be selected for testing and which types of tests should be applied? With tests,
this will be bioburden testing and, in some cases, endotoxin analysis. For these tests,
appropriate alert and action limits should be in place, appropriate to the specific process step.
For sterile products, it is expected that a bioburden reduction occurs throughout the process.
This reduction in bioburden could, for example, be 500 CFU /mL for the start of the process,
moving to 100 CFU/mL for mid-stage formulation, to 10 CFU/mL for later process, and
finally <1 CFU/mL at the point of final filtration (in the case of aseptically filled products).
With endotoxin, the limit prescribed will be based on risk. However, a limit similar to that
used for pharmaceutical grade water (typically 0.25 EU/mL) will often be suitable. These
limits could serve as action levels. The establishment of alert levels is also important.
The levels can be defined as:
Alert Level: A level that, when exceeded, indicates a process may have drifted from
its normal operating condition. Alert levels constitute a warning, but do not
necessarily warrant corrective action.
Action Level: A level that, when exceeded, indicates a process has drifted from its
normal operating range. A response to such an excursion should involve a
documented investigation and corrective action.
When setting 'alert' and 'action' limits, it is good practice to:
Base levels on historical data.
Perform continued trend analysis and data evaluation to determine if the established
levels remain appropriate.
Watch for periodic spikes, even if averages stay within levels.
Consideration should be given to sample containers (sterile, with no leaching or inhibitory
substances.) Containers used for endotoxin sampling must be also be pyrogen-free and made
of a material that does not interfere with the recovery of endotoxin (e.g. glass, polystyrene).
Sample handling (aseptic technique) is also important, as are the storage conditions (samples
placed in 2-8oC within a validated process time); and the assigned expiry time (which should
also be validated.) To add to this, the sample test method must be established and undertaken
consistently. Samples should be mixed in a standardized way prior to testing. Thus a
comprehensive in-process sampling and testing plan is needed for the monitoring of the
manufacturing processes.
Testing will apply to held intermediate product, and here representative samples should be
taken at the end of the hold period. The process validation section below considers the notion
of ‘representativeness’. In addition to the product, excipients and formulated buffers should
also be considered for testing.
For filtered buffers, the testing regime need not be applied to the process at all times. Here it
is recommended that bioburden testing be performed on an appropriate number of batches at
scale, based on statistical analysis and/or risk assessment. Once several buffers have been
tested in conjunction with batches, the test regime can be reduced. For final
diafiltration/formulation buffers, endotoxin testing is additionally recommended.
For buffers that are not filtered these should be freshly prepared and used as soon as possible.
Bioburden and endotoxin testing for non-filtered buffers is generally performed for all
batches at the time of use, for those buffers that have a direct product contact. With buffers
that are prepared immediately prior to use or which are not considered (or proven) to be
growth promoting, a case could be made to exclude them. Certainly with buffers stored for
longer than 24 hours, a hold time validation (including bioburden and endotoxin data) should
be conducted.
The biocontamination control strategy will also inform about process control. Any results
relating to a process hold that are out of limits should be investigated and a drift in trends,
where counts are rising, should prompt a review of the initial assessment. An investigation
must consider the product impact and the origin of the contamination. Once the likely origin
is established, preventative measures should be put in place.
When conducting out of limits investigations, the following can be considered:
Numbers and types of routine bioburden trends (product and environment)
Identification of recovered microorganisms
Evaluation of microorganism for resistance to the sterilization process
Production personnel impact (e.g. proper training or new personnel)
Manufacturing process changes
Sampling and testing procedures changes
Evaluation of laboratory controls and monitors
Additional testing
Cleaning and disinfection of production areas
Any modifications to the sampling plan or changes to operator techniques.
Any raw materials and supplier changes
Origin of contamination, such as water-source contamination or from incoming
materials.
The above list is not intended to be exhaustive.
Process validation
Based on the microbiological risks above, the hold conditions (including both time and
temperature) for a process should be validated to control and prevent potential microbial
growth. These times should relate to the intermediate product and to any buffers or prepared
ingredients intended to be added into the product (14).
The validation plan should consider which time point sampling takes place (in relation to the
process). Here the objective should be at the end of the hold time. According to Clontz:
"Typically as part of a product hold-time validation study, bioburden/ microbial limit testing
is performed at time zero and then at the end of the storage period." (15) Furthermore, it is
important that the sample taken for testing is representative of the product and that it is
homogenous. For this latter reason, some organisations elect to undertake hold time
assessment in conjunction with mixing studies.
In evaluating results, any observed variability between time zero (T0) and the maximum hold
time evaluated (Tmax) should be assessed. The most important aspect is with any rise in
bioburden above either the action level or an acceptance threshold (which may be a certain
increase in bioburden, such as 50%.)
In undertaking validation, not every buffer or excipient will require testing (although it is
good practice to assess each intermediate product stage.) With buffers, it may be possible to
group similar buffers together. Adopting such a matrix approach can save time, although care
needs to be taken with the grouping. It is important to consider the active ingredients, to
determine if they are potentially growth promoting. This will include sugars, which will
promote the growth of many microorganisms; and inherently anti-microbial, such as
hydrochloric acid.
Samples presented for validation should be materials obtained from production-scale batches
and held at set temperatures and times, as defined in process definitions.
Sample taking
The process validation should also consider if the sample can be taken aseptically into a
sterile container. To guard against the risk of adventitious contamination occuring through
sampling, disposable systems that are closed to the environment (like biocontainer bags) will
probably be more suitable than sampling containers. Where individual containers are used,
sampling assemblies with equipment interfaces must either have sampling valves fitted of a
sanitary design or they will need to be disinfected or steamed prior to the sample being taken.
Personnel performing sampling operations must be trained in aseptic sampling techniques.
The manipulation of bioburden samples should be minimized as much as possible prior to
delivery to the microbiology laboratory for testing. Samples are recommended to be stored at
2-8ºC and tested within 24 hours of collection. With the time between sampling and
laboratory testing, this should also be evaluated. The maximum time permitted for sample
transfer from production and testing need be defined. This is an additional variable that
should be controlled to minimize variability.
For sample validation, samples can either be drawn directly from the process or, alternatively,
low-volume bioprocess bags can be used where smaller quantities of the product can be made
up. Such bags are need to be commercially available, and manufactured from the same
materials of construction and contact-surface layers.
Test methods
The test methods, for both bioburden and endotoxin, should be verified as being acceptable
using suitable test methods. Pharmacopeial methods can be used or alternative rapid methods
selected. When selecting non-pharmacopoeial methods, the chosen methods must be
validated.
With bioburden testing either membrane filtration (using a 100 mL sample) or pour plate
(using a 1 mL sample), are the most common methods deployed (16). With both it is
commonplace to select a general purpose agar, such as soyabean casein digest medium
(SCDM), and to incubate samples between 20 and 35oC).
With the verification of established methods, in the case of bioburden, the sample must be
shown to be inhibitory to microbial growth (so that a false negative result is avoided.) This
may involve challenging portions of the sample with a suitable panel of microorganisms, with
challenge inoculums of <100 CFU. The test panel should be made up from microorganisms
traceable to a recognised culture collection; to this, a suitable panel can be enhanced with
environmental isolates. The culture media used and incubation conditions (time and
temperature) must be appropriate (17).
With endotoxin testing, the sample must be shown not to cause inhibition or enhancement
(based on an endotoxin challenge) and where dilution is required, the Maximum Valid
Dilution (MVD) must be not be exceeded (based on the relationship between the endotoxin
limit and the end point of the standard curve or lysate sensitivity.) (18)
Once assessment has been completed, maximum hold times can be established.
Summary
This article has discussed the implications of the process hold times, during pharmaceutical
manufacture, on microbial growth. As the article has described, as risk exists, especially with
biological products for should microbial contamination occur, where microorganisms enter a
product in sufficient numbers, then if the process hold time is long enough and the material
contains sufficient nutrients, then the hold time may be problematic. This can be minimised
as an issue if sufficient controls are in place to prevent contamination occuring, and this
should feed into a biocontamination control strategy. However, to understand the process and
what might occur, the hold time should be assessed as part of process validation. Knowing at
what time point something awry could take place, allows process to be streamlined and for
new controls to be put in place. Monitoring, for both bioburden and bacterial endotoxin, then
provides an assessment of how tight contamination control is and how well controls are
working.
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