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December 13, 2017

Bioburden entry points in downstream processing

By Anastasia G. Lolas, M.Sc., President and Founder of Visionary Pharma Consulting LLC, former FDA reviewer and inspector

If you work in bioprocessing, you are no doubt aware of the need to prevent bioburden entry and proliferation, and reduce the complexity of the equipment and processes involved. Anastasia Lolas, former FDA Biologics License Application (BLA) reviewer and inspector focusing on microbiology, shares her expert tips.


To the extent that a downstream purification train is not a closed system, there is a potential risk of bioburden entry at any point where the system is open to the environment and human operation, or even to other parts of the system. Furthermore, parts of the bioprocessing system that cannot be easily sanitized or sterilized by steam may provide ideal conditions where microbes can hide and proliferate. Prevention of bioburden contamination requires proper procedures and training, and constant vigilance.

Filtration is the first line of defense

Filtration of the process stream entering downstream processing is the first line of defense. The process stream should be filtered before entering the chromatography column, and before entering the in-process pool hold vessel. Monitor the in-process pools for bioburden closely, as they can provide an ideal environment for microorganism growth.

  • TIP: Establish and validate maximum hold times not only for the in-process pools, but also for buffer solutions and any aqueous solutions being used in the process.

The usual suspects

Valves, gaskets and connections are vulnerable to both bioburden entry and proliferation. Further, they are difficult to clean, making proliferation more likely if microorganisms do enter the system. Valves, in particular, are subject to wear and leaking.

  • TIP: When planning maintenance procedures and frequency, take gasket materials and conditions of use into account.

Consider your entire supply chain

Microorganisms can hitchhike a ride on anything, from raw materials to columns and resins to filtration skids. The more handling involved, such as column packing and connections or transfers, the more opportunities for contamination. Vendor and materials qualification is baseline, but it may not be enough. Quality must be an ongoing process, not an event.

  • TIP: Incorporate ongoing testing and quality evaluation for all materials, and periodic review of vendor qualifications into your standard operating procedures.

Don’t forget to evaluate your storage practices and conditions, which can contribute to bioburden proliferation. Storage conditions and containers for packed columns, unpacked resins and cassettes must be validated. Containers that are not dedicated must undergo a validated cleaning process.

Special situations require special care

  • Are you bringing in used skids or columns from a contract manufacturer?
  • Are you transferring equipment between facilities?
  • Are you restarting production following an extended shutdown?
  • Has invasive maintenance been performed on your system?

Those are all circumstances that can increase the risk for bioburden. Protect equipment during transfers and shutdowns, and take special care with cleaning and testing prior to use to ensure that your equipment is not contaminated.

Personnel interface

Any time people interact with purification processes, and especially during open operations, there is potential for bioburden contamination through connections, and exposure of the product to the environment. Training all staff in established procedures and proper technique is a basic requirement. Where possible, design procedures with the mindset of minimizing contamination risks.

  • TIP: Where product is exposed to the environment, make connections under a laminar flow hood or under clean room conditions, especially during manual bulk fill operations. Better yet, design processes with closed systems and connections.
  • TIP: Perform media simulations to test open manual bulk fill operations where the drug substance is filled into multiple containers from a vessel.

Simulations can confirm that proper aseptic technique is implemented, and can reveal weak points in processes and procedures. Simulations are also a useful tool for training new personnel and qualifying personnel in new procedures.

Is your cleaning really cleaning?

Cleaning can be the most effective weapon against bioburden. It is also the primary reason for bioburden in downstream operations.

Water for injection (WFI) is typically used for cleaning purification and filtration equipment, as well as for column packing. Take a holistic view of water system maintenance: delivery hoses, connections, and any storage vessels are also part of the system. Don’t allow the water system to become a pathway for entry of bioburden into your bioprocess stream.

  • TIP: Define time limits for intermediate WFI storage, and filter WFI (through a 0.2µm filter) to avoid microbial proliferation, if WFI will be stored outside of the WFI system in vessels or bags to aid in manufacturing operations (e.g., during column packing and unpacking operations).

Beyond water quality, there are many factors that can determine the effectiveness of your cleaning processes:

  • Contact times
  • Flows
  • Concentration of cleaning agent
  • Dirty and clean hold times

The key question is, can your cleaning processes successfully remove all residues? Unfortunately, packed columns, resins, filters and diafiltration membranes are very difficult to clean. By design, they have large surface areas and pores that are difficult to reach and cannot be steam cleaned. Additionally, most Protein A resins cannot withstand high concentrations of cleaning agents. As equipment ages and campaigns become longer, the original validated cleaning cycle may no longer be adequate.

  • TIP: Designing a robust cleaning regimen and implementing a program to periodically verify the adequacy of your cleaning regimens will be well worth the effort.

Bioburden is an ongoing challenge for downstream processing, but much can be done to minimize the risks. With well-trained personnel, robust procedures and constant vigilance, you will go a long way towards meeting this challenge.

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