Bioburden - Question and Answers on

Biopharmaceutical Manufacturing

Bioburden control now plays a role in various areas of pharmaceutical and

biopharmaceutical manufacturing and quality control. On the one hand, the chapter
<1115> "Bioburden Control of Nonsterile Drug Substances and Products" of the USP
brings this topic of bioburden into focus for drug manufacturers and focuses on the
control of microbial populations throughout the production cycle of excipients, active
ingredients and finished drugs.

On the other hand, "bioburden" is also a sterile manufacturing issue. Annex 1 of the
European GMP Guide requires: "Bioburden should be monitored prior to sterilisation.
Working limits for contamination immediately prior to sterilisation should be established,
based on the efficiency of the process to be used. The bioburden test should be performed
for each batch, both for aseptically filled products and for terminally sterilised products."

Thirdly, bioburden testing for medical devices manufactured or used in the US is

regulated by Title 21 of the Code of Federal Regulations and globally by ISO 11737.

Scope
Based on this the ECA Academy had taken up this topic in a special workshop session to
look at it from different angles and provide information on the legal background as well
as practical examples and strategies for bioburden control. Pharmacopoeia experts,
pharmaceutical quality control representatives and testing laboratories compiled key
information and highlighted the challenges of bioburden control strategy and how to
implement adequate control in companies. From the questions that arose during this
workshop, the experts compiled a first Q&A collection. This first Q&A concerns
"Biopharmaceutical Manufacturing", compiled by the Chair of the ECA Pharmaceutical
Microbiology Working Group, Sven Deutschmann, Roche Diagnostics, and Sebastian
Thoelken from Novartis Pharma Stein AG. In this news you will find a first extract from
this Q&A document, which covers the following areas:

• Bioburden testing - guidelines and regulations

• Microbial control strategy for biopharmaceutical production
• Bioburden for sterile operations
• Evaluation of bioburden excursions in non-sterile biological manufacturing
processes

1. Microbial Control System

• Questions:
(i) "What is the lower specification for a bioburden? A bioburden with
specification less than 1 is not realistic? (due to the environment, consumables
and equipment of the test)? So what should be the lower specification? Less than
3? Less than 5?"
(ii) "Regarding bioburden limits: Samples from BDS production in Class D, C and
B are taken aseptically and analyzed in a non-classified microbiological
laboratory. Occasionally, a couple of colonies can appear on plates from sampling
or handling. Does it make sense to have a bioburden limit of <1 CFU/10 mL in
this setting or what would you recommend?"
Answer: A stepwise progression of limits is defined. Tighter limits are set closer
to the end of the process with ? 10 CFU/10 mL if DS is frozen.

• Questions:
(i) "Did you perform a bioburden method suitability test for each defined
sampling points? Or did you adopt an approach based on a worst case sample
(probability of the intermediate / buffer interferes with the test) to cover some
other steps and reduce method suitability test effort based on a risk
assessment?"
(ii) "Authorities and inspectors - to what extend do they expect monitoring the
control of bioburden in the complete buffer/drug substance/drug product
process, is this all risk based?"
Answer: Yes, a Method Suitability Test is performed for each defined sampling
point. A risk-based approach is used to define the sampling points considering
e.g. amongst others (i) configuration of process equipment, including the
placement of bioburden reduction filters to avoid possible blind spots in
detection of contaminants or (ii) open processing steps and surrounding
environment or (iii) the potential impact of conditioning steps (e.g., extreme pH
adjustments or solvent/detergent additions) for potential inactivation
of putative bioburden must be considered or (iv) the growth-promoting
capability of the process pool.

• Question: "Should buffers which are received sterile filtered be tested for
bioburden before being used in manufacturing? We already test for endotoxin."
Answer: There are no specifications that require this. If sterility of the buffer is
mandatory, then this should also be verified by certificates or demonstrated by
testing.

• Questions:
(i) "Should there be alert levels for all bioburden IPCs taken from aseptic
production?"
(ii) "For aseptic production in class C-D clean rooms, do you still recommend
alert levels for all IPC steps? Or is alert levels for IPC steps only required when
aseptic production in class A-B?"
Answer: Yes.

• Question: "Can processes of pH manipulation of the pool (like viral inactivation)

mask the presence of bioburden or endotoxins in it? I mean, is possible BB being
detected in the BB analysis in a sample of the pool prior to execute the viral
 inactivation and not after the viral inactivation is ended?"
Answer: Yes - the potential impact of conditioning steps (e.g., extreme pH
adjustments or solvent/detergent additions) for potential inactivation of
putative bioburden must be considered.

• Question: "Do you always sample and test for endotoxins in parallel to
Bioburden? If not why?" (note: explicit question to the Novartis colleague)
Answer: Not always but in most cases. Note: EMA GUIDELINE ON THE
STERILISATION OF THE MEDICINAL PRODUCT, ACTIVE SUBSTANCE, EXCIPIENT
AND PRIMARY CONTAINER does not request to test for endotoxins prior to
sterile filtration. Only a bioburden control is requested.

• Question: "If alert levels are required for all IPC steps does it then apply to all
product phases (Phase I-III, PPQ and commercial)? And how do you establish
alert levels for phase I-III and PPQ if only a small amount of batches have been
produced?"
Answer: Yes. Provisional limits are used during development until sufficient
historical data has been generated. Alternatively, for products manufactured
infrequently (e.g. in development) data from similar processes may be used.

• Question: "As the Bioburden is sampled before sterilization by filtration the

product is not yet sterile so some inspectors want us to apply all expectations
that are rather applicable to biological DS. Is this relevant? What would you
recommend?"
Answer: The philosophy of stepwise progression of the limits should be applied
for DP manufacturing as well. Hence, the limits applied for Drug Product
manufacturing should not be less stringent as the limits defined for Drug
Substance. Stepwise progression of limits.

• Question: "What about the bioburden (and its by-products) impact on used
production equipment. In case of microbial counts; there is any
guideline/rationale to assess resins/UFDF membranes safety to be used again in
the manufacturing process? If the event is TNTC and no calculations can be
performed, there is any way (e.g. some kind of blank run study) to defend no
impact due to this byproduct?"
Answer: Cleaning and sanitizing operations should be stringent enough to
remove contaminants and any by-products. The effectiveness of the cleaning and
sanitizing measures must be proven in a cleaning validation and a monitoring of
the cleaning and sanitizing measures must be established in the routine.

• Question: "Regarding hold time limits for manufacturing, is it important to

define hold times for each process step? Or the overall hold time (all process
steps until start of sterilization) is what matters from a microbiological point of
view?"
Answer: Yes. A maximum hold time for each process step must be defined and
validated

• Question: "You described controls to assess the risk of contamination for

Pharmaceutical ingredients and API manufacturers. To your knowledge, is there
 a requirement for environmental monitoring of facilities manufacturing API or
raw material intended to be used for the manufacturing of non-sterile drug
product (dry forms)?"
Answer: If a hygiene zone is defined for the production in which the production
is to take place, then the requirements for this hygiene zone must be fulfilled.

https://www.gmp-compliance.org/gmp-news/bioburden-question-and-answers-on-
biopharmaceutical-manufacturing