Help! The Particle Count Is Overwhelming Us!

By Herman & Erich Bozenhardt

We've noticed a number of FDA Form 483 notices and contamination issues globally, so we felt it's time to address the problem of particulate and microbial contamination directly.

The large scope of contaminations we are seeing lately is the direct result of operational demands, financial pressures, and use of legacy facilities without sufficient modernization. However, any facility can face these challenges and get itself into a deviation, a serious investigation, and potentially a batch rejection.

This discussion will focus only on the contamination burden inside of EU grade D, C, and B manufacturing suites, outside of and only as background to restricted access barrier systems (RABS) or isolator environments.

We often talk loosely about contamination, particle counts, microbial counts, mold hits, bioburden, and other plagues in facilities and processes. The reality is always a very harsh one:

• All these causes are always interrelated.
• They are caused by our people, equipment, and facilities.
• The situation will never resolve itself and never magically disappear.
• If the data shows the problem has abated with no action on your part, that is only temporary, and the problem will come back, often worse.
• Mold in itself is an additional problem, as the source if internal to the facility is a health hazard.

Let’s look at this problem step by step.

Viables Or Non-viables?

When a contamination crisis occurs, typically, we first need to resolve whether the particle is microbial, soil material, or metal particles.

Microbial — Viable

If the particles are bacterial, mold, or fungal, then action needs to be taken to eliminate the source and, more importantly, arrest the proliferation. We will not tackle all the sanitization methods here, but refer to our earlier article, “Cleaning, Sanitizing, Sterilizing, Or Wishing It Away: What Are We Doing To Control Bioburden?”

Inert Soil or Organic — Non-viable

Soil and organic particles pose a problem through product contamination and entrainment into the product. Although we may not instinctively tackle this like microbial contamination, we should, because most of these infiltrates will be a nutrient source for microbials and accelerate microbial contamination shortly thereafter.

Metal — Non-viable

These are easier to identify by looking at the pattern of particle generation, location of the particles, and the size. Rotating equipment that is wearing sheds metal particles (generally in a range of 0.5 to 25 microns). Reciprocating equipment like filling line equipment sheds particles from nearly invisible metal to metal contact (smaller than 0.5 microns). They all must be examined within proximity of the peak of the particle data. In these cases, we should perform continuous monitoring and we will quickly see various patterns of:

• Continuous level – then ask, what motor is on all the time?
• Square wave or sawtooth pattern – matches with the usage of certain equipment or transfer/material handling gear.
• Continuous ramp — metal-to-metal abrasion is continuous and the HVAC around it is not removing it; to the contrary, it is retaining it in an aerial vortex.

Where Does It Come From?

Microbial particles, dirt, and everything else in the class of exterior contaminations come from the exterior of our non-classified areas into our classified areas and find their way via our normal avenues of process flow. That is the key: the “normal flow” of the process, which could be weigh/dispense, material transfers, equipment movement, gown in/gown out, and often the facility itself.

People

Consistently, people and their gowning practices are one of the largest sources of contaminants in the classified area due to the entrainment of dirt, soil, and debris on the shoes, and the lack of proper facility finishes, architectural features, and gowning practices to eliminate the carryover. In most organizations, operators travel from the street into a non-classified area and then into a large locker room. Soil is retained on the soles of shoes, then transferred to their socks in the locker room, then into the CNC grade D gowning rooms. This practice often carries the contaminants on the booties of the operators and then with the constant “on and off” bootie exchange shakes the contaminates back on the floors, and on the operators’ hands while doing the procedure.

The locker room soil issue will continue until the facility implements dedicated shoes, cleaned, and sanitized daily, inside a CNC/D room and disposable socks or a bootie strategy that really works when implemented.

The second overall problem with people-based particles is the gowning practices and the use of hands over the clean gown, gowns touching the floor, and the rapid sequence of gowning. Bi-directional gowning/ungowning rooms exacerbate this problem in older facilities.

Pallets

We have concluded that moving pallets containing raw materials, glass, RTUs, supplies, etc., is the single greatest supply route for microbials and particles into an EU grade D or C environment. First, if you are reading this and have wooden pallets in any part of the production facility, including hallways and non-classified spaces, except the warehouse or outer areas, you have a serious problem and are dealing with the single greatest contaminant source in the industry. There must be no wood pallets inside a biopharma facility.

The normal pallets used in the biopharma business are polymer-based (white or blue), steel, or aluminum. Polymer pallets, while much cleaner than wood, have many interstitial spaces, corners, and holes where contaminants will reside.

Pallets, in general, travel from the filthiest warehouse to a vendor’s parking lot, to the back of a truck, and eventually into your classified area. Even your warehouse is not clean enough. Due to the height of the pallet and the size of interior spaces, polymer pallets can’t be adequately cleaned by the operators, and the constant movement over the dirtiest areas of any plant section makes it the greatest source of contamination proliferation.

Equipment

Equipment moving from suite to suite, in and out of cleaning areas, through water puddles, and across classifications, which may also include NC areas, will collect dirt, micros, and debris on the wheels, in the wheel bearings, under the carriage, and on the vessel body. This is most common with tanks, carts, and skids. We have yet to see a practical method to clean some of the vessels. We use wipe-down and spray-in techniques, but rarely are the methods completely effective. The decontamination of the equipment may be effective, but it leaves the material airlock (MAL) as the key contamination repository from the cleaning effort. If you consider an excellent equipment wipe down in the MAL, the contaminants removed from the equipment are now on the floor of the MAL and on the operators themselves.

As we clean and sanitize over the months of use, the steel surfaces of equipment eventually become rouged or rusted. These surfaces harbor mostly anaerobic microorganisms and make it nearly impossible to clean them effectively. In addition, the rouge constantly sheds metal particles simply by contact.

Relative to equipment, we should not lose track of the metal particles as the source of the non-viable contamination. Setup changes, especially hurried setup changes on packaging equipment are generally a source. All too often our setup mechanics adopt poor habits as the time pressure drives their practices. A careful review of the setup, by the equipment manufacturer is the ideal remediation. Review of all the setup alignments, measurement recheck, and physical review, and photographic comparison is needed. Robots and their sensors often need calibration. A misaligned laser or camera could cause a robot or automated material handling system to abrade metal to metal. Expert help, measurements and detailed histories are key here.

Materials and Material Containers

These items are of varying sizes and configurations and include various containers, sacks, and drums, which may not be as cleanable as needed. Their geometry serves as a contaminant reservoir. Secondly, these materials travel around the facility and in many cases come directly from vendor warehouses. Their undersides are never cleaned, and they contribute to the contamination load in the MALs and inside classified suites.

Facility

Even the building itself contributes its own piece to the contamination puzzle. We have covered the problems with legacy facilities (architectural finishes, layouts, and construction) in BioProcess Online before; please see these past articles, which cover the challenges and the solutions.

• "Bioprocess Facility Design — Layout Rules and Configurations"
• "Remediation, Renovation, Reconstruction for Aseptic Facilities: When Does It All End?"

Fixing It all

No single aspect covers the single root cause because it usually takes several flaws in the facility and process in concert with one another to cause a contamination outbreak.

As an example, with high plant demands, a specific MAL sees a high level of traffic with warehouse deliveries many times during a shift. This can be compounded with carts and pallets blocking the MAL’s HVAC return. Sanitization frequency of the MAL may not have been properly addressed because of staffing shortages or the frequency simply fails to match traffic levels. We can now see how an interior grade C suite could be experiencing several high mold counts as the traffic, lack of cleaning, and congestion creates a vicious cycle. This is how it starts, and the end point is undetermined until we tackle several of the following remediation steps:

Pallets

• Pallets are easy to solve with a little effort. First, buy cleanroom pallets, which are either a solid polymer with no interior crevices or solid aluminum.
• Invest in a hot water/steam cleaning pallet washer with a constant fresh water replenishment. Too many legacy pallet washers recirculate dirty water.
• When pallets are washed, shroud groups of them in a polymer wrap and move the wrapped pallet bundle to CNC/cleaner spaces for manufacturing use.
• Dedicate a specific number of cleanroom pallets for use only in designated CNC, D and C, and B spaces. Exchange materials at MALs so pallets do not move around the plant. Within each EU grade space, operations personnel are responsible for cleaning the pallets as a normal routine.
• Build a pallet exchange barrier in all the MALs to assure that cleanroom pallets never move from a lower classification area into a higher classification area. This can be implemented with an 8-inch-high barrier, 6 inches wide, and placed across the MAL, which compels operators to push or pull materials across the barrier, from pallet to pallet, onto the cleaner pallet on the way in. Likewise, materials move by design from the higher grade to the lower grade pallet on the way out.

People and Gowning

As much as one would think, changes in gowning require changes in approach and behavior. The following are some very practical steps to eliminate the contamination ingress:

• Dedicated shoes are a key step in the process, but only if the dedicated shoes are held in the grade CNC transition room to a grade D gowning room, under air circulation, UV lights, and/or sanitized daily.
• For high-traffic locker rooms, implementing a disposable sock or bootie transition from the locker room to the CNC to grade D prevents operators from bringing in soil from the locker room. This can be implemented easiest with one-way changing booths (with in and out doors) and by eliminating traditional lockers.
• Unidirectional flow of personnel airlocks (PALs) with separate gowning and degowning rooms.
• Eliminate the abuse of gowns via gown dragging and minimizing gown contact with the floor during the gowning and ungowning process. This is a training and practice measure. 
• The gowning room layout should have a unidirectional flow and easy-to-clean floors, floor level return HVAC registers, and a high air change rate.
• The gowning sequence optimizes the gown-in process and forces frequent sanitizing steps. This should include long-sleeve scrubs and a grade C head covering that is one piece and covers the top of the head through the neck.

Cleaning up the MALs

Incoming MALs naturally will pick up the greatest number of contaminants, so they require our most serious attention, along with pallets.

First, use polymer materials for walls and ceilings and epoxy terrazzo for the floors to make them durable and highly cleanable. Supplement cleaning with UV lights overhead or UV integrated in the LED lighting system. This will help reduce microbial counts.

"HEPA helpers" or stand-alone integrated HEPA/UV circulation systems supplement the typically poor HVAC inside the MAL. These can bring dramatic benefits by trapping contaminants from the air and the floor in filter media.

Finally, cleaning needs to happen at high frequency inside incoming MALs. We recommend sanitizing the walls, floors, and ceilings during every shift. Remember, mold sticks to ceilings.

Unidirectional flow, with in-only and out-only MALs is the easiest way to prevent contamination concentration and proliferation, and in do so comply with best cGMP practices.

Don’t Move Those Tanks

Frequently in legacy facilities, we see suites and even buildings sharing equipment. This, by its nature, invites contamination transfer from one area to the next.  The contamination on the vessel body plus the exposure of any threaded fittings and the micro loading of the metallurgical surface make this a dangerous process.

As a basic premise, just don't do it.

Strongly consider using single-use disposable systems (SUS) and have those function as the tanks with fixed pallet tanks in the suites, route process flow through disposable tubing systems, and use aseptic quick connects for vessel-to-vessel transfer without moving equipment.

You also might consider moving the entire process into one suite. Last, if you can't get away from moving tanks, use a floor-level pass-through (instead of a traditional MAL) with a gaseous decontamination cycle.

Transfer Systems

In order to deal with legacy facilities and protect the product during transfer, all process transfers must be closed. The product is most vulnerable when many ingredients need to be added into a vessel, especially powders. Open-hatch transfers can be closed by delivering the ingredients with a closed single-use bag, like ILC DoverPacs, with a single or double butterfly valve and a polymer tri-clamp fitting that connects to the receiving vessel. In that case, we are not exposing the outside of the container to the open vessel and not aspirating materials in the dumping exercise.

Cleaning Up the Facility

The final area we need to cover is actually very simple. It involves actively upgrading the architectural finishes of the facility and removing the greatest vulnerabilities:

• Review the flooring in the material, personnel, and product transfer path.  Seal all floor cracks and chips and recoat the floor so dirt and micros do not make it a contamination reservoir.
• Scale pits have a vast volume of uncleanable area with an open drain at the bottom open to a sewer. This provides a wealth of spore-generating organisms. Remove all weigh scale pits, fill them in, and replace them with modern electronic low-profile scales that can be lifted or swung up for cleaning.
• Drains need to be closed with a heavy metal gasketed cover when not in use. Just before closing the drain, bleach or another anti-microbial should be added.
• Sprinklers need to be cleanroom recessed sprinklers. The old industrial sprinklers are uncleanable. Yes, some facilities still have them.
• Threaded connections do not belong anywhere in a GMP facility. This not only is valid for process or product lines, but for utility lines, conduits, and instrument connections. Threaded connections on all process lines need to be changed to sanitary fittings, and non-process threaded connections need to be covered with stretch film to make the entire line cleanable.
• Eye wash and safety showers tend to be invisible to most of us; however, at specific frequency, these units are tested, which requires the release of domestic (rust- and micro-laden) water into the classified area. These tests are generally ignored by operations, but they need to be controlled, contained, and shrouded because they could release more contaminants into the area than all the other sources we discussed. Safety showers should be located or relocated out of the direct process area. After each test, the area needs to be dried, sanitized, and an anti-microbial solution needs to be poured down the shower drain. Today, eye wash stations and safety showers are being replaced across the board with single-use polymer pressurized saline stations. They can be bought through most catalog companies in various sizes and volumes. These systems do not require testing, and the solution is noncontaminating.

Conclusion

Due to the recent surge in biopharm production and the poorly planned use of legacy facilities we have encountered a serious number of recent contamination stories. Our facilities will always be challenged by contaminations but recognizing the source or sources must lead to remediation. In this article we have developed several high probability scenario sources and discussed how to remediate them.

About The Authors:

Herman Bozenhardt has 46 years of experience in pharmaceutical, biotechnology, medical device manufacturing, engineering, and compliance. He is a recognized expert in the area of aseptic filling facilities and systems and has extensive experience in the manufacture of therapeutic biologicals and vaccines. His current consulting work focuses on the areas of aseptic systems, biological manufacturing, and automation/computer systems. He has a B.S. in chemical engineering and an M.S. in system engineering, both from the Polytechnic Institute of Brooklyn, now New York University. Reach him by email at hermanbozenhardt@gmail.com and on LinkedIn.

Erich Bozenhardt, PE, is the lead process engineer for regenerative medicine operations at United Therapeutics. He has 16 years of experience in the biotechnology and aseptic processing business and has led several biological manufacturing projects, including cell therapies, mammalian cell culture, and novel delivery systems. He has a B.S. in chemical engineering and an MBA, both from the University of Delaware.