GUIDE TO INSPECTIONS OF VIRAL CLEARANCE PROCESSES
FOR PLASMA DERIVATIVES TABLE OF CONTENTS
Scope and Intent . . Pg 1
Introduction . . Pg 1
Background . . Pg 2
Scope of the Risks . . Pg 2
Methods of Viral Clearance . . Pg 3
Validation of Viral Clearance . . Pg 4
Operations and Inspections . . Pg 6
Removal Methods . . Pg 7
Inactivation Methods. . .Pg 7
Additional Considerations. . .Pg 7
Conclusions. . .Pg 8
SCOPE AND INTENT
The intended audience for this document is
the ORA field investigator. The document's
origins stem from the transfer of primary
responsibility for GMP inspections of
fractionators from CBER to ORA in the latter
part of 1996. While FDA field investigators
are well versed in the application of GMP to
the drug industry, this document is intended to
communicate differences in the application of
these regulations occasioned by the uniqueness
of plasma derivative products.
In particular, GMP requires and ORA field
investigators determine through inspections,
that significant aspects of drug manufacturing
operations have been validated. Although the
fractionation industry shares many of the same
process validation concerns with the larger
sterile drug manufacturing community, the
validation of viral clearance processes presents
issues with which the field investigator may not
be familiar.
This document, which has been prepared
jointly by the Office of Regulatory Affairs
(ORA) and the Center for Biologics Evaluation
and Research (CBER), attempts to provide
basic information on viral clearance validation
concepts, to enable the investigator to
distinguish those areas of viral clearance
processes that are appropriate for GMP
inspectional coverage. It is not an
all-encompassing technical guide on viral
inactivation processes, but rather a document
that seeks to assist field investigators in
conducting fractionator inspections. If the need
arises, this document may be amended or
expanded to include other issues of interest to
the field investigator concerning viral clearance
processes.
This guidance represents the Agency's
current thinking regarding inspectional
considerations of viral clearance processes for
plasma derivatives.
INTRODUCTION
Plasma-derived proteins are important
therapeutics for many patients in the U.S. every
year. Because they are manufactured from
human plasma, special precautions must be
taken during the production of these proteins
to assure against the possibility of the products
transmitting infectious diseases to the
recipients.
This document focuses on the possibility
that a small proportion of the individual units of
plasma used as source material may be
contaminated with human viruses and the
processes used to remove or inactivate those
viruses. Additionally, as is the case for any
pharmaceutical, there is a possibility of
introducing adventitious pathogens during
production. This risk is one specifically
addressed by adherence to current Good
Manufacturing Practices (GMP) and for this
reason it is a natural focus of any facility
inspection. This document is to be used in
conjunction with the Compliance Program for
the inspection of Plasma Derivatives of Human
Origin (7342.006), the Guideline on the
General Principles of Process Validation (May
1987), the Investigations Operations Manual
(IOM), and the Code of Federal Regulations,
Title 21 (21 CFR).
BACKGROUND
Addressing the risks that may be associated
with plasma derivatives begins with the plasma
from which the products are manufactured. A
number of precautions are taken to prevent
contaminated units from being collected and to
eliminate contaminated units when they are
collected. These now familiar precautions
include:
1. Screening donors by medical and behavioral
risk history;
2. Maintaining donor deferral registries to
prevent the use of units from unsuitable
donors;
3. Testing blood and plasma donations for
markers of disease;
4. Quarantining blood and plasma until tests
and control procedures establish its safety;
and,
5. Monitoring and investigating adverse
incidents to ensure that deficiencies are
corrected.
Many investigators will be familiar with these
procedures from their inspections of blood
banks and plasma collection facilities. When
the plasma is used as source material for the
manufacture of plasma derivatives, an additional
layer of safety may be achieved by including
effective viral clearance step(s) in the
manufacturing process.
SCOPE OF THE RISKS
The viruses of greatest concern for safety of
plasma derivatives include the hepatitis viruses
(hepatitis B and C, abbreviated HBV and HCV,
respectively) and the human immunodeficiency
viruses Types 1 and 2 (collectively HIV; the
causative agent of AIDS). Another virus,
human parvovirus B19 (B19), is a common
contaminant of plasma. Hepatitis A virus (HAV)
contaminates plasma products less frequently.
B19 and HAV pose some unique problems
because they are small (15-30 nm) and do not
possess an outer envelope composed of lipids
(both are termed "non-enveloped" viruses for
this reason). Both B19 and HAV are
extraordinarily resistant to heat and chemical
treatment and are difficult to remove by
nanofiltration. Furthermore, no licensed
screening kits exist in the U.S. for either virus.
Fortunately, the diseases caused by B19 and
HAV are less serious than for other viruses of
concern, although they may cause serious
disease in susceptable populations.
Nevertheless, there are intense efforts
underway to develop new methods for
removing or inactivating these viruses and some
of these methods may begin to appear in
manufacturing processes over the next few
years.
The most clinically significant viruses, HIV,
HBV and HCV, are all "enveloped" viruses,
which renders them more susceptible to
inactivation methods based on chemical
treatments or heat. These viruses are also
larger and more effectively removed by
nanofiltration. Nearly all plasma derivative
manufacturing processes now include effective
methods for their inactivation and/or removal.
It should be noted that certain viruses which
may pose serious risks when associated with
blood components (human T-lymphotropic
virus (HTLV-I & -II) or cytomegalovirus (CMV))
are not considered to be significant risks with
respect to plasma derivatives. These viruses
infect white cells and therefore are associated
with the cellular components of blood which
are mostly absent from plasma. The viruses
also tend to lose their viability over periods of
refrigerated storage and they do not tolerate
freezing and thawing. For this reason, HTLV-I
& -II and CMV are not risk factors for plasma
derivatives.
It should also be noted that the possibility
that bacteria or protozoa may contaminate a
unit of plasma or whole blood is not
considered a significant risk to recipients of
plasma derivatives. Manufacturing under GMP
should remove or inactivate any of these
endogenous agents, and any exogenous agents
introduced during manufacture of the drug
substance. Therefore, properly processed
plasma derivatives pose no additional risk of
bacterial or protozoal contamination beyond
those associated with any aseptically processed
parenteral. However, the processes must be
carefully evaluated to insure that they
consistently produce sterile products ( see
Compliance Program 7342.006 and the
"Guideline on Sterile Drug Products Produced
by Aseptic Processing" 1987).
This document also does not cover possible
risks posed by the poorly characterized agents
thought to be responsible for transmissible
spongiform encephalopathies. There are no
known effective methods for screening out
plasma units that contain the infectious agent,
and no known manufacturing methods that
would be effective in removing or inactivating
the infectious agent.
Finally, this document applies to human
plasma derived products only. Products
derived from animal plasma pose different risks
than human plasma derivatives. Bovine plasma,
for example, may not be sourced from
countries in which the disease, bovine
spongiform encephalopathy (BSE) exists (FR
94-21279). Otherwise, no formal
recommendations regarding manufacturing
safeguards for TSEs have been developed at this
time.
METHODS OF VIRAL CLEARANCE
A number of different types of
manufacturing steps are capable of removing or
inactivating viruses that may be present in
plasma pools from source or recovered plasma
donations. These steps may be divided into
two broad categories: removal, or partitioning,
is the physical separation of the virus or viral
particles from the therapeutic component; and
inactivation, which destroys the virus so that
the remaining viral fragments lack the structure
and components needed to infect an individual
receiving the product.
Removal processes include filtration, affinity
chromatography, ion exchange chromatography,
and polyethylene glycol fractionation. Heating
and solvent detergent treatments are examples
of processes that inactivate viruses.
Additionally, some processes, such as ethanol
fractionation, both remove and inactivate
viruses. Finally, a number of new methods are
under development, such as irradiation,
photoinactivation and treatment with a variety
of chemicals. Several of these novel techniques
have been incorporated into the production of
investigational products. In order to be
effective, viral inactivation techniques must
destroy at least one viral element essential to
replication. Photosensitizing techniques use
light-activated dyes that are irradiated, causing
the dyes to convert to molecules that can alter
DNA or membrane lipoproteins. Heat
treatment denatures viral proteins and nucleic
acids, rendering viruses incapable of replication.
Irradiation processes may destroy viral nucleic
acids by inducing breaks and linkages. Solvent
detergent techniques destroy the viral envelope
in lipid-enveloped viruses.
VALIDATION OF VIRAL CLEARANCE
All processes which claim to remove virus
must be fully validated by the manufacturer, as
there are no methods codified in the applicable
regulations. All of these methods are employed
during production of the drug substance.
A specific viral inactivation method that is
employed on the drug product in final
containers, is heat-treatment at an attained
temperature of 60 C ñ 0.5 C for 10-11
(continuous) hours. This process is mandated
for two products: Albumin (Human) and Plasma
Protein Fraction (Human) in 21 CFR 640.81
and 640.91, respectively. Because the
conditions of treatment are specified by
regulation, manufacturers are not required to
validate the effectiveness of the treatment itself,
but must nevertheless demonstrate that the
process operating parameters are met
consistently during production.
Some other products are heat treated, in
bulk tanks or containers, using similar
methodology (e.g. Antihemophilic Factor
(Human), Intravenous Immune Globulin
(Human)); alternative methods may also be
used, such as vapor heat treatment of
lyophilized intermediates. Another inactivation
method that may be encountered (e.g. Immune
Globulin Intravenous (Human) is treatment of
in-process material with solvent and detergent,
for a given time at a given temperature, and
subsequent removal of these additives. This
removal is generally accomplished by extraction
and/or the use of chromatographic methods. In
all of these cases, it is the responsibility of the
manufacturer to validate the effectiveness of
the method, as well as demonstrate that the
validated operating parameters are met
consistently during production.
Validation studies are performed when a
new product is under development or when a
manufacturer wishes to introduce a new step
into a pre-existing manufacturing process. In
these cases, the validation is designed along
with the process which may be optimized for
viral clearance. It is necessary to consider
scale-up problems and the possibility that
validation studies at the pilot scale may not be
relevant to the final manufacturing process. If
scale-up involves changes in the manufacturing
process that could adversely affect the viral
clearance validated at the pilot stage, then viral
clearance would have to be revalidated for the
altered process.
It is also possible that a manufacturer, with a
product made by a well established process,
identifies a step in that process which may be
effective in removing or inactivating viruses.
The manufacturer may then attempt to validate
this step without changing it. In this case, the
manufacturer should take the process as it
exists, without attempting to optimize it for
viral clearance, in order to avoid additional
studies to demonstrate that the product has
not been adversely affected. If the process has
changed, the manufacturer should demonstrate
that the final product has not been adversely
affected.
Regardless of the setting in which the
validation is performed, all viral clearance
validation studies share certain common
features. These can be summarized as follows.
1. Validation is performed on scaled down
laboratory models of the production
process. It is an unacceptable hazard to
introduce viruses into a production area in
order to validate a viral clearance step. It is
also impossible in most cases to produce
sufficient amounts of a virus to validate its
removal at the full manufacturing scale. The
necessity of working with scaled down
systems presents discreet challenges to
validation of viral clearance processes:
a. The scaled down model should
accurately represent the full scale
manufacturing process. This is insured
by controlling parameters such as
temperature, volume, flow rates, contact
times, relative geometries, and load. The
most important measure is the actual
performance of the scaled down process,
measured by parameters such as
capacity, yield and purification if
applicable.
b. The starting material (process
intermediate) should accurately reflect
that of the full scale manufacturing
process. This is often accomplished by
sampling process material from a full
scale batch.
c. Even though scaled down systems are
used for validation, the studies should be
designed to represent the worst case
conditions that could be encountered
during full-scale production, i.e., those
conditions under which the removal or
inactivation of viruses would be expected
to be the least effective. The viral
titrations should be designed with
adequate replicates to assure a
scientifically and statistically sound result.
Validation of the scale-down model
should involve multiple runs of the
model, the results of which are
compared to those from the full-scale
process.
d. Appropriate operational and
performance qualification with reference
to the critical operating parameters
should be performed for the full-scale
manufacturing system. Qualification
should include acquiring sufficient data to
verify that the full-scale system
consistently delivers all critical
performance conditions as was specified
in the successful validation studies with
the representative scale-down system.
e. Subsequent changes in the full scale
manufacturing process can affect the
validity of prior viral clearance studies.
Whenever a process and/or equipment
change is made, the possible effect of
that change on viral clearance should be
considered and evaluated, and the viral
clearance should be revalidated to the
extent necessary.
2. Viral clearance validation studies usually take
the form of "spiking" experiments, in which
large amounts of a virus is added to the test
article. It should be noted that the viral
load is far in excess of what would be
expected in a "contaminated" plasma pool.
The reduction in the amount of added virus
by the manufacturing step in question is then
measured. Appropriate controls are
included to insure that the measurement of
the amounts (titers) of the virus does not
change the performance of the scaled-down
manufacturing step. Additional controls are
included to confirm viability of the indicator
cells and the infectivity of the virus.
3. In vitro analyses are most commonly used to
quantify virus levels in the course of a
validation study. These may take the form
of "plaque assays" or assays that measure
"cytopathic effect", both of which are
performed in tissue culture. These assays
measure infectivity of the virus used in the
study. Biochemical assays may also be
encountered, such as antigen-based or
nucleic acid assays (e.g., PCR). These are
acceptable when predictive of infectivity.
Finally, some studies may use animal models
such as primates or ducks, but these have
become less frequent because they are
expensive and adequate alternative methods
are available.
4. Most validation studies are performed with
model viruses sharing characteristics of the
relevant human viruses. The selection of
appropriate models is of critical importance.
Among the human viruses of concern, only
HIV and HAV have appropriate in vitro
systems with which their titers can be
measured. The clearance of HBV and HCV,
by some manufacturing processes, has been
validated in primate models, but these
studies are not common today.
5. The extent of validation with respect to the
number of steps validated and the number of
different viruses studied varies greatly from
product to product and from manufacturer
to manufacturer. With the exception of
some IGIM products (see footnote 4, page
8), all plasma derivatives are subjected to at
least one viral clearance step, in addition to
the fractionation process itself which has
been shown to reduce viral load for some
agents.
6. A validation study may be acceptable even if
some detectable virus is found, as
these studies should be designed to add
many times more virus to the test article
than would be encountered in actual
practice. Large amounts of virus ("high
titers") permit more precise quantification
and provide safety margins to the
manufacturing process.
7. When more than one manufacturing step in
a process has been validated to clear a
particular virus, the overall clearance of the
process is the product of the two steps
(often calculated by adding log reduction
factors), provided that the two steps
operate by independent principles. For
instance, the results of a filtration step and a
heating step may be combined because they
operate by different mechanisms. On the
other hand, merely repeating a step would
not double the viral clearance because there
would be no second mechanism involved; an
additive effect cannot be presumed.
8. Validation of the process or processes
serves to establish that the operating
parameters, used during normal production,
are appropriate. Changes may be made
based on the validation study. It is therefore
important that the parameters established
during the study are those used in actual
manufacture.
OPERATIONS AND INSPECTIONS
The validation of viral clearance methods is a
highly sophisticated and scientifically complex
undertaking. From time to time, it is expected
that technical questions will arise during the
inspection. The investigator should keep in
mind that the scientific resources of the Agency
are at his/her disposal. Questions may be
referred at any time during the inspection to
the Inspections Task Force (ITF) in CBER's
Office of Compliance (301-827-6191). The ITF
representative will contact the appropriate
reviewer to provide information.
The following elements are suggested as
important aspects of inspecting a manufacturing
process from the point of view of viral safety.
1. In most cases, the validation studies have
undergone scientific review, or are in the
process of being reviewed by CBER staff, as
part of the license application. As such, a
detailed technical review by the
investigatorof the findings is generally not
necessary. However, the manufacturer
should be following the process exactly, as
approved or submitted for approval, and the
investigators should familiarize themselves
with the approved and pending procedures
in applications and supplements. As an
alternative, studies which support claims of
viral safety, either approved by or pending
approval by CBER should be available for
review in the manufacturer's license files,
on-site, for the product(s) in question.
2. Validation studies may have been conducted
by the manufacturer, but often the studies
are performed by a contract firm that
specializes in such studies. As such, the raw
data for the viral assays may not be available
at the licensed site. Available records may
consist of reports from the contractor.
Audits of these contract sites should be
performed periodically to review and verify
raw data generated during these, and other,
studies. Data available at the licensed site
should also be reviewed for integrity.
3. Examine the full-scale manufacturing step
itself to insure that it consistently meets the
critical validated operating parameters that
were defined in the viral validation study. In
addition, manufacturing records should be
reviewed to insure that the process is
carried out as intended. Some examples
follow:
Removal Methods:
a) Removal methods involving nanofiltration
are fairly new in the industry. The
membranes are generally single use
involving tangential flow. Insure that:
i. validated filtration parameters are
being followed, e.g. pressure and
flow rates; and
ii. the membrane is post-production
integrity tested. (NOTE: unlike
membranes used for microbial
retention, for some of these
membranes this is accomplished
using colloids such as dextran.
These tests may be destructive.
Forward flow and bubble point
methods are not applicable with
these membranes).
b) Chromatographic methods (e.g. affinity,
ion exchange) are used in purification
of many plasma derivatives. These
methods may also be validated for their
viral removal capabilities. Insure that:
i. validated parameters (e.g. flow
rates, column height, protein load)
are being consistently followed;
ii. if a validated number of uses has
been established for the column
resin(s), the firm is consistently
using the resin(s) for no more than
the validated number of uses
(NOTE: Often, concurrent
validation is allowed to establish an
acceptable number of times for use
of the resin. If this is the case,
in-process parameters should be
consistently monitored to insure
column performance. In addition,
the resins continued ability to
remove validated levels of virus
should be evaluated, periodically,
during the concurrent study); and
iii. regeneration procedures are
followed between each lot of
product.
Inactivation Methods:
a) Heat treatment processes, either
codified in the regulations or validated
by the manufacturer, should be
performed according to pre-established
parameters to insure effectiveness.
Insure that:
i. the proper time and temperature
parameters are met consistently;
ii. the equipment used is qualified,
maintained and monitored to
insure that these parameters are
met; and
iii. if problems occur, e.g., the process
is interrupted, the manufacturer
should not "repeat" the process
unless such repetition has been
validated and approved under the
license to demonstrate that the
additional heat treatment does not
adversely affect the properties of
the product.
b) Manufacturers who use
Solvent/Detergent methods must
demonstrate that the validated
parameters are met both during the
treatment and the subsequent removal
of the additives used. Insure that:
i. the proper concentrations of
additives are consistently used, and
treatment occurs for the validated
time.
ii. removal procedures are being
properly followed (e.g. If a column
is used, it is employed for the
validated number of uses or
monitored for performance (see
NOTE section 3 (b)(ii)); and
iii. the in-process drug substance is
tested to insure that the additives
have been removed to validated
levels.
Additional Considerations:
1. Inspect the Adverse Event Report/complaint
files and the procedures the company
has developed for handling them. Companies
are required to report incidents of
suspected transmission of viruses to FDA in
30 day periodic reports.
2. Establish that the firm has not changed steps
upstream of the validated viral removal step
without the proper reporting to FDA. This
may significantly change the characteristics of
the intermediate entering the viral
removal/inactivation step(s) and should be
submitted to the Agency per 21 CFR 601.12.
3. Insure that there has been no change in the
ordering of steps in the manufacturing
process. This may affect the effectiveness of
the viral inactivation/removal steps. These
changes also should be reported as specified
in 21 CFR 601.12.
For your information, sanitizing agents
(bacteriocidal and/or sporicidal) used in the
cleaning of the facility and equipment generally
serve to inactivate the viruses of concern for
plasma derivatives (refer to the Compliance
Program 7342.006 for further instruction on
inspection of cleaning of facilities and
equipment).
CONCLUSIONS
The establishment inspection provides a
critical element in assuring the viral safety of
plasma derivatives. Although scientifically
sound methods for removing or inactivating
viral contaminants may have been designed into
a production process, it is essential that these
methods be executed consistently and reliably
on a day-to-day and lot-to-lot basis in order to
provide assurance that the product is safe for
use. The establishment inspection is the
principle setting in which the operational
validity of viral clearance procedures may be