Introduction

Aseptic filling of sterile drugs, also know as sterile filling, still remains one of the most critical
processes in biopharmaceutical manufacturing. This is due to its highly technique driven processes
and the potential safety impact to the end user, usually an already compromised patient. There are
only indirect safeguards for the sterility of the filled drug after it is stoppered and capped in the
cleanroom.
Unlike terminal sterilized filled drugs, the stability of the aseptic filled drugs will be affected by
steam autoclave, dry heat ovens, Ethylene Oxide, and irradiation, either Cobalt 60 Gamma or E
Beam. Thus the need to utilize an aseptic process to fill certain biologicals, pharmaceuticals and
biotechnology drugs.
The history of aseptic fill/finish processing is relatively recent with the sterility requirements for
injectables being established in the 1920s and large scale biological manufacturing of blood and
plasma products during WWII. Plasma products did have, and some products still use, a post-fill
pasteurization process of low heat treatment of 60C for 10 hours. Pasteurization does not provide
sterility, but can reduce the contamination of fungi. Anti-fungicidal reagents were also added to
parenteral drugs to help mitigate the contamination that was occurring with early aseptic
processing.
Then in an effort to help improve consistency in aseptic processing, the Parenteral Drug Association
(PDA) published its Aseptic Validation Technical Report in 1981 [8]. This was followed by the Food
& Drug Administration (FDA) in 1987 with its Aseptic Processing Guidelines [1]. The International
Society of Pharmaceutical Engineering (ISPE) published its Sterile Facilities as part of their Guidelines
Series in 1999 [14]. Recently, the FDA published its Concept Paper: Aseptic Guidelines in 2003 [15].
Aseptic filling is an aseptic process that requires the close coordination and complex interaction
between personnel, sterilized product, the fill/finish equipment system, cleanroom and support
facilities, and sterilized filling components.
There is also the perception issue for aseptic fill/finish, which is another reason for the many
safeguards that I will discuss shortly, since micro contamination is not readily visible. Micro
contamination is very small, and the surfaces that look clean and sterile may in fact not be. Thus the
aseptic fill/finish processes are highly dependent on technique, detailed procedures, equipment and
controls.
Regulatory Considerations

As with our industry, there are many global regulatory requirements for aseptic/ sterile fill/finish
manufacturing. Although each country or geography has its regulatory guidance, we have not yet
achieved full harmonization. Most of these are listed in this articles appendix, and I will be only
briefly discussing the current FDA 1987 Guidance. This FDA Guidance provides a couple of nice
definitions for us.
In aseptic processing, the drug product, container and closure are subjected to sterilization
processes separately and then brought together Because there is no further processing to sterilize
the product after it is in its final container; it is critical to the maintenance of product sterility that
containers be filled and closed in an environment of extremelv high quality
We also have written in the Code of Federal Regulation (CFR), section 21 CFR 211.113 (b) that states
Appropriate written procedures, designed to prevent microbiological contamination of drug
products purporting to be sterile, shall be established and followed. Such procedures shall include
validation of any sterilization processes.
Another section, 21 CFR 211.167 (a) states
For each batch of drug product purporting to be sterile and/or pyrogen-free, there shall be
appropriate laboratory testing to determine conformance to such requirements. The test procedure
shall be in writing and shall be followed.
Currently, the FDA has been expressing a number of concerns about aseptic manufacturing, citing
all drugs recalled due to non-sterility over the last 10 years were produced by aseptic processing
(Spring 2002). If you drill down in these recalls, you will find that there are a few companies who
have multiple recalls, and that there are a lot of documentation recalls. These are situations in
which the documentation or procedures had omissions and errors and as a result a recall was
initiated. The consensus within our industry is that, in fact, we have been getting much better with
our aseptic filling processes

Aseptic Fill/Finish

What can be aseptically filled? Virtually any solution, powder or suspension that can be terminally
sterilized prior to the aseptic fill/finish process. Typically sterile drugs are aseptic fill/finish in molded
glass bottles, tubular glass vials, tubular glass syringes and in Europe more than the United States,
glass ampoules. Solutions can also be subsequently lyophilized in a sterile dryer to further stabilize
drugs. The more unique the product or container system, the greater the technical or operational
challenges that may ensue.
How do we complete the aseptic fill/finish process? You need to decontaminate the operational
personnel, terminally sterilize the drug product, filling components, equipment change parts and
sanitize the cleanroom and in-place equipment. Then bring it all together with good aseptic
practices, and the simplified process maps look like the aseptic process map.
The aseptic fill/finish methods can vary between an early clinical phase hand fill (clinical solution fill
photo), to small volume semi-automated filling to the fully automated high-volume over multiple
day production batches.
Cleanroom Personnel
The personnel in the cleanroom are like the double-edged sword, they are absolutely necessary to
complete the aseptic fill/finish process, but at the same time, provide the greatest microbial risk for
a sterile product. You are constantly regenerating yourself, and in the process shedding a huge
amount of dead skin and other particles. An average person is capable of shedding ten million
particles per day. That is further compounded by the types of clothes worn and where you have
recently been, such as what might be on the bottom of your shoes right now.
Thus the amount of investment that is made to limit the operational personnel risk to the sterile
drug. Personnel are encapsulated with extensive sterile gowns and qualified gowning procedures.
The cleanrooms have extensive unidirectional flow air currents to provide a barrier as well as sweep
the potential contamination away from the exposed drugs.
Automated fill/ finish equipment is installed to reduce the amount of personnel who are present in
the cleanroom during the aseptic fill/finish processing. Development of the current barrier
equipment designs and the recent development of the isolator technology have been made to
further isolate the exposed sterile drug from the operational personnel.
Lastly, the implementation of Best Aseptic Practices to provide personnel with methods, training and
qualified procedures to further prevent microbial contamination of the sterile drugs.
The Best Aseptic Practices are a set of best practice methods for personnel to govem themselves as
they move and function in the cleanroom environment while executing their processes. Aseptic
practices will also incorporate the specific requirements for your aseptic fill/finish processing.
Procedures are needed for both gowning and de-gowning processes, and state hygiene, training,
qualification and re-qualification requirements.
Sterile outer garments are usually made of synthetic or natural materials, worn as an outer garment,
which have low or no particle shedding or penetration characteristics. Most companies outsource
their sterile garment preparation to a company who will wash and sterilize their garments, usually
sterilize with Gamma. For low volume sterile garmenting requirements, you can utilize single-use
sterile garment packs. The sterile outer garments act as a personnel filter to isolate the individual
and their contaminants from the cleanroom environment and the sterile drugs.
Personnel who function in the aseptic fill/finish aseptic processing core will need to have completed
a gowning qualification, especially to be present in the clean room core during a sterile fill
operation. This would include the operational personnel, maintenance mechanics, quality assurance
and quality control personnel, production management, engineers and technicians. The qualification
should include training on the basics of microbiology and the Best Aseptic Practices. Typically, this is
followed by a gowning demonstration, then a gowning critique of the person in training.
Final gowning qualification should be completed with multiple sterile gowning in the cleanroom
with microbial testing inside the cleanroom. I recommend that the sterile gowning and microbial
events should be videotaped to provide the operator with additional feedback and assist with the
analysis of the gowning techniques. Gown qualification best practices require the gowning
qualification to pass three consecutive microbial testing and successful media participation prior to
being deemed gowning qualified. An example of a gowning process is provided in Table 2.
Facilities: The Cleanroom
The cleanrooms are controlled areas and in conjunction with the supporting utility systems and
facility infrastructure, create the environmental envelop in which the aseptic fill/finish process
operates. As with the other components of the aseptic processing, the cleanrooms area complex
combination of physical rooms and areas, utilizing High Efficiency Particulate Air (HEPA) to create
unidirectional air patterns, maintenance of positive pressure between rooms in conjunction with
constant air changes, and sanitization processes. All of this operates with constant environmental
monitoring (EM).
The cleanroom design will take into consideration the flow of personnel, product, equipment and
components. Effective aseptic fill/ finish facility designs will take into account the flow of all of these
from the receipt of raw materials at the warehouse through the facility to final warehousing. A very
simplified illustration is the cleanroom cascade defense.
Very basic to the cleanroom design is the classification of the cleanrooms relative to the operation
that is occurring within it as, well as adjacent to it. Harmonization of the regulatory guidelines for
cleanrooms has not fully occurred yet, but I believe we are making some progress. In the cleanroom
classification table (Table 3) is a very simplified comparison between the European Annex l and FDA
classifications. I have referenced the various cleanroom compliance documents in the article
appendix, and an in-depth discussion of cleanroom classifications was not intended for this article.
You will need to know where your products are going to be distributed to select the proper
guidance to follow, which for our industry and global products, usually means all of them.

Before discussing the cleanroom materials of construction or the Heating, Ventilation and Air
Condition (HVAC), it is critical to first understand the flow of personnel, sterilized components and
sterile product in developing the cleanroom design and operation. The flow requirements may vary
with each sterile drug produced.
The personnel flow, as I discussed earlier, is very critical to maintaining the sterile environment. This
would include the gowning, degowning and all of the necessary movements through all of the
cleanroom facilities. It is ideal to ensure that the personnel flow is moving one-way; from gowning
to operation and then degowning, cleanest area towards the dirtiest.
The one-way movement within the cleanroom, especially the sterile core for the aseptic fill/finish
operation, is critical for all of the material, products and components. You will want to ensure your
cleanroom design will eliminate two-way transfers from occurring concurrently, where sterile goods
are physically passing non-sterile goods where there is a potential for microbial cross-
contamination.
The movement of mobile tanks with sterile filter bulk drug presents challenges as well, as the
exterior surfaces cannot be terminally sterilized with the drug enclosed before the aseptic fill/finish
operation. The bulk tanks will require sanitization in airlocks or at other transfer modules. The
sanitization processes for mobile tanks are challenged by the amount of fixtures on the tanks,
clearance under the tank, and the tank wheel assemblies. Frequently the mobile tanks are
segregated from the aseptic core and only the transfer of the bulk tank tubing connection
necessary for the aseptic connection.
The equipment layout and flow will also influence the cleanroom design. The ideal aseptic fill/ finish
system is a fully automated in-line isolator fill/finish system. The most difficult to manage and
presenting the greater microbial risk, is a batch sterilization and completely manual filling process
that occurs in a bio-hazard safety hood.
The equipment flow should also reflect the necessary sterilized set-up parts that will be changed for
each sterile drug batch, such as the filling needles, stopper bowl and feeder components. The
sterilized set-up components may require a specialized technician or mechanic to support the
aseptic fill/finish operation. The ease in which the aseptic set-up can be accomplished and
maintained can directly influence the quality of the aseptic fill/finish operation. You should eliminate
any operations that require a sterile operator to reach over the fill line.
The aseptic core in which the sterile drug is actually exposed to the cleanroom environment is the
most crucial area of a cleanroom, and warrants the most detailed attention to the design of the
cleanroom. This is the area where the sterile drug is transferred from the filling needles to the
sterile container. Typically the stoppering or closing of the container occurs immediately after, with
the exception of when the drug requires sterile lyophilization. The requirements of the lyophilization
process require the stopper be only partially seated on the vial.
For solution drugs after a stoppering process, sealing occurs immediately, usually with some kind of
aluminium seal. The design of the cleanroom or equipment would include a barrier between the
stoppering and sealing processes to minimize any potential aluminium contamination.
Another variation for sterile drug solutions is the use of Form-Fill-Seal (F-F-S). This fully automated
process forms a plastic container system, filling the solution during the process and immediately
sealing the containers. The F-F-S process minimizes the environmental exposure and provides
microbial contamination results similar to an isolator process.
For lyophilized drugs, the filled and partially stoppered vials would be transferred to a sterile
lyophilizer (drier) for the completion of the lyophilization cycle. It is normal for the stoppers to be
seated in the vials inside the sterile drier at the end of the lyophilization cycle prior to opening the
door. The stoppered vials are then removed from the sterile drier and immediately capped. The
delay in sealing the container, immediately after the filling process, allows the drug to be exposed
to the environment is an additional risk that occurs with sterile lyophilization.
An essential component to the cleanroom is the Heating, Ventilation and Air Condition (HVAC)
systems. The HVAC systems that support pharmaceutical operations, especially cleanrooms and
aseptic manufacturing, are complex and extensive. The heating and cooling functions are needed for
operator comfort and environmental control. Ventilation function provides the necessary circulation
and air turns to maintain environmental control. The HVAC will also be designed and operated to
maintain the aseptic core by the use of positive pressure that extends away from the core.
A rule of thumb for temperature in the heavily gowned aseptic core is mid 60F, and the relative
humidity above 30% to control static, and below 60% to control rust, organism growth and
personnel from sweating.
For cleanrooms, the ducts terminate at High Efficiency Particulate Air (HEPA) filters. For the HVAC
that supports the aseptic processing operations, including the preparations area, there will be a
series of pre-filters prior to the HEPA filters. The HEPA filters are rated 99.95% effective for microbial
retention and facilitate unidirectional air flow. Previously, it was thought that a laminar air flow
pattern could be effectively achieved with the HEPA filters, but with the knowledge gained by
extensive smoke studies of class 100 aseptic cores, the more realistic expectation is a unidirectional
air flow pattern.
The HEPA filters are the achilles heel of the cleanroom HVAC system. They require extensive care
and maintenance and could have a detrimental effect on the quality of the cleanroom environment
if not well maintained. HEPA filters have the potential to fail within the filter medium, at the gaskets,
seals and frame.

Other utilities that are needed to support the aseptic fill/finish operation include Water for Injection
(WFI), oil-less compressed air, nitrogen gas, sterile steam and vacuum. The compressed air and
nitrogen gas will also have point of use sterile filters inside the aseptic core, and the vacuum system
should have one-way check valves. The WF I is predominately used in the preparations for the
rinsing of vials, stoppers and equipment change parts. The intent of this article was not to provide
an overview of the utility design and operation that support cleanroom operations.
Materials of construction of a cleanroom should facilitate the required operation, which includes
extensive cleaning processes and support the required environment control. The surfaces should be
hard, smooth and easily cleanable. The floors, ceiling and walls should be continuous, with flush
installations and utilizing welded joints where possible. The wall fixtures should be flush mounted to
surfaces and the lighting fixtures flush mounted and preferably remote access. Surfaces should not
be designed to allow the build up of particulate contamination.
Most aseptic cleanrooms have telecommunication equipment to allow discussions without the need
of personnel leaving and entering the operation. Increasingly, video monitoring and recording
cameras are installed in the aseptic core. The video equipment allows a further reduction of
monitoring personnel inside the critical area, where each additional person incrementally increases
the risk of microbial contamination.
Cleanroom maintenance and sanitization requires the qualification of the cleaning and disinfectants
agents. The qualification of the sanitization processes will need to be done in conjunction with a
documented process and trained personnel. This qualification should include the development of
the expiration dates for the formulated sanitization solutions.
Some of the common disinfectants and sterilants are phenolics, sterile alcohol, hydrogen peroxide,
Quaternary Ammonium, Sodium Hypochlorite, and Formalin. Phenolics will work with organic
matter, but have issues with resistant spores. Alcohols will act quickly, but have little effect on
spores and are flammable (e.g. 70% Isopropyl Alcohol, IPA). Hydrogen Peroxide is an excellent
sporicidal, but also not compatible with all surface agents (e.g. soap). Quaternary Ammonium
(Quarts) are highly stable and nontoxic, but affected by water quality, soap, and not effective with
spores. Formalin is effective against bacteria spores, will not corrode metal, but it is toxic with
irritating fumes and residuals that can be difficult to clean. Sodium Hypochlorite is an excellent
sporicidal but affects metals.
The cleanroom sanitization process requires full sterile gowning and all of the required aseptic
techniques that would be utilized during the aseptic filling. As with the aseptic filling process, the
cleanroom sanitization process requires documentation, personnel training and qualification.
Environmental Monitoring (EM) is the process to ensure that the cleanroom is under control for
potential viable and non-viable contamination. Your EM process should have qualified
methodologies to routinely collect, evaluate and interpret EM data. The determination of sampling
points and required limits should be defined in your documentation. Your EM program should
identify periods of critical activity where sterile product may be exposed to environmental
conditions (photo Em class 100 Bio Safety Hood).
Sterile Product Filtration
Your drug will require sterilization by some method prior to the aseptic filling process. Traditionally,
the bulk drug sterilization is accomplished by filtration, normally a depth filter. You will need to bulk
drug a method for sterilization and a sterile container system that is compatible with the drug and
your aseptic fill/finish process. The drugs can be pre-sterile filtered (e.g. .45 micron), followed by a
series of at least two sterile filters at .22 micron. The sterile filters are both pre- and post-bubble
tested to ensure integrity. The Sterile bulk is then transferred to the aseptic fill and aseptically
connected to the fill equipment. Currently the best-in-class for sterile filtration is a closed system
that extends from the non-sterile bulk to the aseptic filling equipment.
Filling Components

All components and supplies that are required during the aseptic fill/finish operation must be either
sterilized or sanitized. Sterilization is usually completed with pass-through steam autoclaves, dry-
heat oven or tunnel and sanitized cleanroom airlocks.
The filling components can also vary per the drug and production requirements. Tubular Type I
glass vials are predominately used for Small Volume Parenterals (SVP) and sterile lyophilization and
blown Type II glass bottles for Large Volume Parenterals (LVP). Tubular Type I glass stock is also
predominately used for aseptic syringe production. A number of manufacturers are considering
Cyclic Olefin Copolymer (COC) vials that function similar to glass vials.
The stoppering of the vial provides the sterile seal of the drug from the environment and a crimp-
seal cap ensures the long term integrity. The stopper also provides a barrier to gas and oxygen to
the drug ensuring long term stability. Elastomeric closures (stoppers) that are used for parenteral
solutions are formulated to ensure product stability and patient functionality. Two of the basic styles
of closures are the plug for sterile solutions and the leg for sterile lyophilization (clinical solution
fill photo). Some of the considerations should be given to size, type and number of needle
punctures, water vapor transmission rate, ability to retain bound water, gas transmission, stoppering
equipment of the filling line and potential extractables.
Component Preparation
There are many ways to aseptically fill/finish sterile drugs, which includes the traditional solution
filling of glass vials and syringes, sterile powder fills, sterile lyophilization, and blow-fill-seal. For this
article, I will only be addressing solution filling and sterile lyophilization. There are many
considerations in the selection of your aseptic filling equipment. To name but a few: solution
volume, fill tolerance, production throughput, drug viscosity, drug foaming, gas blanketing, drug
temperature, potent compounds, drug stability and reactivity.
Sterile preparation of the vials and bottles is achieved by rinsing (washing) to remove endotoxins.
The glass vials and bottles are depyrogenation usually with hot air. This is accomplished in a batch
mode with an oven, or a continuous process with a tunnel that connects the bottle washer to the
filling station.
For small parts cleaning, such as filling needles, forceps and stoppering equipment, as well as
stoppers, you will complete the initial washing/rinsing to remove endotoxins and loose particulate.
Then wrap the parts for subsequent steam autoclave processing to destroy the endotoxins.
Depending on the formulation, the stoppers may be able to be sterilized by irradiation.
Aseptic fill/finish processes can very from a clinical hand fill, to semi-automated mono-block, to a
high speed filling lines. Filling equipment systems can be characterized as either Open, Barrier,
Isolator and RABS.
Filling lines are characterized as having no barriers or other physical restrictions between the sterile
operator and the sterile drugs. As a result of EU regulation, open fill lines are not common to
commercial aseptic operation, but can be found in Phase I and II clinical manufacturing operations.
The barrier filling lines have transparent panels that restrict sterile operator access to the sterile
drug. Some of the barrier panels may be designed as doors to the Barrier with very specific
operational procedures that support aseptic techniques for use during aseptic fill/finish production.

The solution filling process will include the transport of sterilized vials and bottles, orientation to the
filling station, a means for check weighing, stoppering and crimping stations. For high speed lines,
there will also be accumulation tables and vial load/loading stations. The filling equipment can
include the sophistication of in-line check weigher, automated vision systems, reject stations, and
SCADA information systems networked from each equipments PLC.
As a rule of thumb, the stoppering and capping (sealing) should be completed as soon as possible.
There is some discussion that the crimping of the cap may not require the same critical
environment as the solution filling process and crimping may be a particulate generating process.
The norm for solution filling equipment is to provide a barrier between the stoppering and capping
processes. Isolator systems are a current alternative to the classic barrier equipment installation.
Isolators utilize a glove box technology and they are designed for minimal human intervention
which provides increased contamination control. A majority of the isolators are sanitized by
vaporized hydrogen peroxide. Isolators require more expensive capital investment, can be more
complex to install, qualify and operate and may have less flexibility to changeover fill sizes and
products. They have historically been designed for high-volume dedicated drug production and
microbiological quality laboratory operations. There is also a trend to utilize Campaigning for
Isolators technology installations [16].
An alternative to isolator technology is the Restricted Access Barrier System (RABS) a term first
described by Upjohn, now Pfizer. RABS is similar to the isolator technology utilizing glove ports and
other sterile operator restrictions. It is also similar to the traditional barrier fill line with the
utilization of a conventional aseptic core cleanroom. The gowning and aseptic techniques are the
same as a barrier fill/finish operation. The advantages that have been reported are reduced capital
investment, quicker validations and operational start-up, reduction in lot-to-lot turn around time.
RABS operations have documented contamination control over a traditional barrier fill/finish system.
Sterile Lyophilization

A sterile lyophilization process requires all of the basics for aseptic processing of a solution product,
but with the additional processing requirements and risks of the sterile dryer (Lyo) equipment.
Sterile dryers are now designed to utilize Clean-in-Place (CIP) and Sterilization-in-Place (SIP) of both
the condenser and the product chamber.
The chamber which holds the drug product being processed requires a loading methodology that is
consistent with aseptic techniques. For high production and large sterile dryers, the majority of new
installations also include automated load and unload equipment. The automated load/unload
capability reduces the headcount inside the aseptic core and should reduce the risk to microbial
contamination.
The lyophilization process includes filling the product solution aseptically, with the stopper partially
seated in the vial. The partially stoppered vial is then transported and loaded into the sterile dryer,
thus the sterile product has an extended exposure to the environment. The drug solution is then
frozen by either immersion in liquid nitrogen prior to loading or by the sterile shelf. The
lyophilization cycle includes the primary and secondary (terminal) drying. After the lyophilization
cycle has been completed, the stoppers are usually seated into the vial by lowering the dryer
shelves. A sterile drug producer may need to stopper the lyophilized vials under vacuum or and
inert gas. Then the dryer door is opened and the stoppered vials are transported to a capping
(crimping) process.
Media fills for process validation for a sterile dryer is not a full process simulation. The lyophilization
process is usually conducted under near vacuum, with a slight amount of pressure provided by
sterile nitrogen and at -35C or colder. All three of these variables have a negative effect on media
and will distort the results. Thus, most companies will modify the media fill in the sterile dryer by
not freezing the shelves, not evacuating the chamber and connecting sterile air to the chamber
inlet.
Visual Packaging Inspection
Visual packaging inspection of aseptic filled drugs is usually completed 14 days after fill. This is a
period of time that could allow the growth of any potential contaminating micro organisms. The
critical inspection process is for the presence of a cloudy or hazy solution that would indicate a
contamination potential. The manual version of this inspection occurs with the use of white and
black background viewing areas.
Manual visual inspection requires trained and tested inspectors, and due to the repetition of the
inspection task, it is really only about 85% effective. Thus a number of companies have
implemented double- inspection of the product, very tight acceptance criteria and automation of
the process with vision systems.