. .
[DOCKti NO. 84N-0154]
DRAFT OF POINTS TO CONS:[DER
: IN THE CJi.ARACTERIZATION OF CELL LINES
USED
“Esubmit.~tiritten.
..
TO PRODUCE. BIOLOGICMS
(1993)
comeqt.s.Q@,
th}.~.tid~~ft
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Submit written requestsdfor
single copies
of this draft to:
.
: ;:
.’.
.
For further ‘infox%%tipn..:
~bout
G
.. .
this dra,ft,
‘“
-..- contact:’
..
.
:.,
,.,
&enter’ for [email protected]
and ,Resealrch(“HFM-20) “= ‘“:’:”
Food and DxWg Administration
‘:
1401 Rockville Pike, Suite ~?OON
20852-1448::,:,”. L ,
Rockville, ~
301-402-3190
.!..
.,
Comments and--requests “should be identified with the docket number.,
found’in.b.rackets $,pj$!leheading’:of
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..>;.
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4
id
‘L
DEPART=T
OF HEALTH & HUMAN
Public Heatth Service
SERVICES
Memorandum
JuL 121993
DATE:
FROM:
Director,
Center for Biologics
SUBJECT:
Points to Consider in the Characterization
to Produce Biological
(1993)
TO:
Manufacturers
Biologics
Utilizing
Evaluation
and Research
of Cell Lines Used
Cell Lines for the Production
of
This Points to Consider Document on the Characterization
of Cell Lines
Used to Produce Biological
(revised May 1993), is intended to replace the
document of the same title issued in 1987. These “Points” are neither
regulations nor guidelines, but serve to represent the current consensus
of the Center for Biologics Evaluation and Research (CBER) staff.
.
We intend to continually revise and update this document as necessary
You are requested to review and
order to improve it’s usefulness.
comment on this document.
All comments should be addressed to:
Dockets Management Branch
Food and C)rug Administration
12420 Parklawn Drive, Room 1-23
Rockville, MD 20857 _
_
Comments
should be identified
by the docket number
Two copies of any comments
should be submitted.
8/11/93
s. alter, HFM-630
Note:
d
84 N-0154.
‘
in
1
POINTS TO CONSIDER IN THE CHARACTEI+ZATION OF CELL LINES
USED TO PRODUCE BIOLOGICAL (1993)
CENTER FOR BIOLOGICS EVALUATIC)N AND RESEARCH
FOOD AND DRUG ADMINIS~TION
Table
1.
INTRODUCTION
3
Il.
HISTORY AND GENERAL CHARACTERISTICS OF THE
CELL LINE
7
Ill.
\
Page
of Contents
Iv.
v.
A.
History
of the Cell Line
B.
General
Characteristics
7
8
of the Cell Line
8
THE CELL BANK SYSTEM
A.
Generation
B.
Storage
c.
Cell
8
of Cell Banks
9
of the Cell Banks
Bank
10
Qualification
PRODUCTION CULIURES AND PRODUCT TESTING
A.
Cell Culture
B.
Management
-
12
12
Media
of Cell Culture:;
14
17
QUALllY CONTROL TESTING
A.
Tests for the Presence
of B;acteria and Fungi
17
B.
Tests for the Presence
ot Mycoplasmas
17
c.
Tests
of Viruses
17
D.
Tumorigenicity
for the Presence
Testing
22
2
V1.
VALIDATION OF VIRAL ELIMINATION
24
A. Design.
26
B.
Interpretation.
30
C.
Statistics
32
D. Revalidation
32
VII .
cONcLLlsloN
32
Vlll.
RHERmcEs
34
lx.
Attachment
#1:
Letter to Manufacturers
Requesting
Identification
of Bovine/Ovine
Components and Sources
36
Recommended
Mycoplasmas
38
1,
x
Attachment
#2:
‘Test Procedures
for
3
1.
INTRODUCTION
This document
produce
Public
is concerned
biological
Health
products
Service
with the characterization
which are subject
of cell lines
to Iicensure
under the U.S.
Act and also with the identification
adventitious
infectious
agents
contaminate
the final product.
from
the cell
lines
The existing
which
general
used to
of possible
might
regulations
in 21 CFR
200 et seq. and 21 .CFR 600 et seq. and especially 21 CFR 610.18 et seq.
embody
requirements
consistent
from
This document
in achieving
with objectives
lot-to-lot
and
provides
these
free
information
objectives,
that thle final
from
product
adventitious
infectious
that may be useful
but does not create
be uniform,
agents.
to manufacturers
new requirements
or
rights.
Advances
in biotechnology
be evaluated
is subject
available.
issues
that
from cell lines should
investigational
product
process
to change
Accordingly,
scientific
license
rapidly.
in light of its own particular
line and manufacturing
document
are occurring
should
who
(PLAs).
product
(INDs)
Existing
findings
in this
become
as raising
products
development
and before
200 series and 600 series, are also broadly relevant
consulted.
information
biological
general
should
and the cell
be interpreted
produce
both during
applications
applications
Therefore,
as new and significant
manufacturers
new drug
characteristics
being used.
this discussion
consider
Each new product
under
submitting
regulations,
21 CFR
and should be
4
These
pOiIIk
suitable
in specific
appropriateness
and certain
of the points
specified
Therefore,
approaches
aspects
the scientific
basis
approaches
the Center for Biologics
will review the adequacy
of testing
may well be
may not be applicable
for determining
for consideration
rapidly and more appropriate
the future.
(CBER)
situations,
Furthermore
to all situations.
developing
Alternative
are not all-inclusive.
here
the
is
may be developed
Evaluation
in
and Research
of any cell line on a case-by-
case basis.
This document
supersedes
Characterization
reflects
approach
focuses
on:
3.
However,
emanating
from’s several
international
biological
and characterization
of the manufacturing
process
and/or
inactivation
testing
of the bulk and final product
of the
of adventitious
for removal
.
agents;
and
to assure
safety.
it should be noted that a number of tests previously
have been revised or eliminated.
Karyology
and
As stalled in the 1987 PTC, the
with cell lines to produce
identification
(1987)”
substrate;
validation
recommended
1.
to working
production,
ce!l
2.
of changes
held since that time (1,2).
current
1.
(PTC) in the
of Cell Lines Used to Produlce Biological
a number
workshops
the “Points to Consider
Specifically:
products
5
In 1978 an ad hoc committee
karyology
(3).
control.
The Committee’s
The detailed
described
diploid
rep~~rt was published
characterization
in 1979 applied
and monitoring
specifically
liowever,
of continuous
karyology
the utility
cell lines
is probably
is not recommended
2. Tumorigenicity
Experience
applied
to
live virus
for the characterization
mirlimal;
therefore,
routine
in these circumstances.
testing
has shown
rodent origin
in 1979
of, for example,
of karyology
on
procedures
and are still
cell lines used for the production
vaccines.
tested
met tO revise the recommendations
that virtually
are tumorigenic;
for live virus vaccine
production
In addition,
tumorigenicity.
cell or gene therapy
therefore,
Human
-
for tumorigenicity.
all ?ontinuous
epithelial
should,
some special
may reqtire
rodent
cells
cells
however,
cases
tumorigenicity
cell
lines
of
need not be
and all cells
be tested
regarding
used
for
somatic
testing.
.
--
3.
Oncogene
Recent studies
cell growth
testing
indicating
suggest
that oncogene:s may be involved
that testing
for endogenous
in normal
oncogenes
is not
necessary.
The results
support
general,
of tests described
the acceptability
testing
should
in this document
may be submitted
of a cell line to pro~duce a biological
be performed
:n compliance
with Good
to
product:
Laboratory
In
6
Practice
requirements
as checklists.
These te:jts should
(21 CFR 58).
Rather, the selection
of tests depends
on many variables
such as the nature of the cell iine, the manufacturing
product
In addition,
indication.
be greater to support approval
The testing
required
and its use.
for initiating
clinical
of testing
may
trials
depends
on the product
of issues each
address the basis
manufacturer
must
when making these decisions.
biological
of a cell line intended
includes:
1.
history
2.
the cell bank system;- and
3.
quality
In addition,
and generaI
control
monoclinal
for use in the manufacture
,.\
characteristics
of the cell
information
antibodies
by the manufacturing
concerning
the testing
and recombinant
and Testing
of Monoclinal
1507)” (now under revision),
process.
of cell lines
DNA technology
Antibc~dy Products
“Points
and Testing of New Drugs and Biological
Technology
line;
studies ~f virus
vivo and select in vitro use may be found in the “Points
Manufacture
of
testing.
in many cases there is need for validation
and inactivation
Additional
(June
and the
that is needed
here do no{ generally
due to the variety
The characterization
removal
situation
of a PI-A than that for an IND application.
The points discussed
for test selection
consider
the amount
not be interpreted
to Consider
used to produce
products
for in
to Consider
for Human Use
in the Production
Produced by Recombinant
(April 1985)” and the supplement
in the
to the recombinant
DNA
DNA
7
Points
to Consider,
(1992).”
Consider
Acid
Characteri;!ation
If a cell !ine or cells are to be returned
its biological
Somatic
‘Nucleic
product(s)
M vhm, then the “Points
Cell Therapy and Gene Therapy
in the Collection,
Mononuclear
Leukocytes
Processing
and Genetic
into humans
Stability
to produce
to Consider
in Human
(199-1)“ and aiso the Points to
and Testing
for Administration
of Ex-Vivo-Activated
to Humans
(1989)
should
be
consulted.
Il.
HISTORY AND GENERAL CHARACTERISTICS OF THE CEU LINE
A.
History
of the Cell Line
<:
The history
products
of any cell line used for th~e production
should, include,
when possible:
1.
age, sex and species of the donor;
2.
for human cell lines, the donor’s
available,
the results
the detection
3.
culture
history
medical
performed
of the tissues
passage
history,
passage
in animals,
previous
identity
on t~
--
and if
donor
for
methods
from which
used
the line was
media used and history
of
etc.;
testing
agent
history
agents;
of the cell line including
derived,
adventitious
of tests
of adventitious
for the isolation
4.
of biological
and the
testing.
results
of all available
8’
B
General
The growth
Characteristics
pattern
and morphological
should be determined
cells
[Points
Characterization
and Genetic
specific
that may be useful
markers
(such as marker
should
finite
be characterized
through
senescence
of the cell line
to Consider:
Stability
chromosomes,
life expectancy,
appearance
and should be stable from the master cell bank
to the end-of-production
Ill.
of the Cell Line
(1992)”].
!f there
in characterizing
specific
surface
number
Acid
are
the cell line
markers),
If the cells
for stability.
the total
“Nucleic
these
have an identified
of population
doubling
levels
should be determined.
\..
THE CEU BANK SYSTEM
A.
Generation
of Cell Banks
Once a cell line is chosen as the biological source of a product,
cell bank system should be generated
supply of equivalent
material,
a detailed
likelihood
the advantages
to providing
and increasing
the detection
would consist
(MCB) and a manufacturer’s
supply of starting
include
allowing
of the cell line and decreasing
and adventitious
cell bank system
a constant
of a cell bank system
characterization
contamination
to assure that an adequate
cells exist for use over the entire -life span of
In addition
the product.
a
for
the
of both cell line cross-
agent contamination.
Ordinarily,
of two tiers: a master
cell bank
working cell bank (MWCB).
the
9
The Master
Cell Bank is defined
composition
derived
as a collection
of cells of uniform
from a single tissue! or cell.
It is Cryopreserved
stored in the liquid or vapor phase of liquid nitrogen.
in aliquots
‘“i he
MCB for a diploid cell line should be pre!pared from cells at a low
population
doubling
The Manufacturer’s
level.
Working Cell Bank (MWCB) is derived
from one or
more ampules- of the tvtCB. The_MCB source cells are expanded
seria! subculture
manufacturer
combined
up to a passage
and approved
number selected
to form the MWCB.
into individual
and
of a lot of a biological
If cells from more than one MWCB ampule are used, the cell
suspensions
doubling
upper
ampules
One o’h more such ampules from
the MWCB would be used for the production
product.
by the
by CBER. At that point the cells are
into one pool, dispensed
cryopreserved
by
should be pooled at the time of thawing.
level of cells used for production
limit based
on written
criteria
should
established
The population
not exceed
an
by the
manufacturer.
B.
Storage
of the Cell Banks
Both the MCB and the MWCB should be stored in either the liquid or
vapor phase of liquid nitrogen.
of individual
ampoules
The location,
identity
of cells should be thoroughly
and inventory
documented.
It
is recommended
that the MCB and MWCB should each be stored in two
or more widely
separate
as at a distant
areas
within
the production
facility
site in order to avoid loss of the cell line.
as well
10
C. Cell Bank Qualification
1.
The Master Cell Bank
Testing to qualify cell banks should be done either on an
aliquot
of the cell bank or on cell cultures
cell bank, as appropriate.
testing
to demonstrate
freedom
identity
testing.
include
tests
Testing
for adventitious
The testing
for bacteria,
and cell culture
line, to detect
which
described
shbuld
tests
agents
agents
mycoplasmas
include
in selected
Finally, testing
to determine
in vivo
specific
tests
of the cell
Some of the
circumstances
should
should
routine
history
viruses.
and
and for viruses.
amd any other
contaminating
are relevant
most circumstances
for adventitious
viruses
from the
the MCB includes
adventitious
based on the passage
possible
in part V.
to qualify
from
fungi,
inoculation
that are warranted,
tests
Testing
derived
are
be performed
ilf the cells
in
produce
b
relroviruses
or retrovirus
particles.
This
testing &
also
described
in part V.
Extensive
identity testing of the MC,B should be done once and
should
include
properties
throughout
should
all tests
needed
to establish
of the cells and the stability
the manufacturing
include:
process.
all significant
of these
Such
properties
characteristics
11
a. morphology,
as determined
by light and electron
microscopy;
b. species
of origin (and sex, if human);
ratio;
c. split
that the cells can be used for
d. data demonstrating
their intended purpose.
expression
derived
If the cells contain
system to produce
a recombinant
protein, data should be obtained
demonstrate
expression
protein it produces
and the quality
DNA-
to
the copy num~er and physical
system
an
state of the
and quantity
( see Points to Consider
of the
on rDNA
products);
e. a meaningful
test should
be performed
for routine
production
f.
cultures
identity
used for each
which
testing
will also
OF
lot of product;
and
other such tests which may be useful for
demonstrating
with
2.
be performed
the
Manufacturers’
that the cell bank
intended
Working
characteristics.
Cell Bank
is comprised
of ceils
12
The MWCB being derived from the MCB and propagated for an
approved
number of passages
in tissue culture,
be spot checked for contaminants
introduced
include
vivo)
from the culture
sterility,
tests
that may have been
medium.
mycoplasma,
only needs to
routine
and cell line authenticity
Recommended
tests
virus
and in
(in vitro
to check
for cell line
cross-contamination.
When a manufacturer
serum
free defined
moves from a serum containing
growth
cells which are weaned
recloned to establish
growth
in the defined
medium,it
is suggested
to a
that the
into the serum free medium should be
a new MCB al~d MWCB of cells for optimal
medium.
IV. PRODUCTION CULTURES AND PRODUCT TESTING
Quality
control
of product
culture
quality
media,
A.
of cell substrates
control.
management
Cell Culture
Accurate
records
cell culture
biological
the manufacturer
is an important
areas to be addressed
of cell cultures,
inclade
and specific
part
cell
testing.
Media
should be kept of the composition
medium.
product
Specific
used for production
and source of the
In cases where the manufacturer
uses a proprietary
medium
of the medium or medium
of a
or medium
supplement
supplement,
may be
13
required
to supply the necessary
for example,
of a Master
derived
cell culture
they should
contaminants
and adventitious
for the production
of Bovine
should be provided
testing
Acceptance
from animal sources
agents,
of certified
raw
manufacturer
accepting
certification
is
carried
to be free from
responsible
Encephalopathy.
Information
and source
out on these
materials
based
of, and
additives.
on certification
should be based on a determination
the product
that ‘the process
by the
used for
sufficient.
Since animal serum may produce
attempts
should
for the propagation
The residual
are added to the
such as the agent
Spongiform
agents
provided by the supplier
subjects,
be certified
with regard to the identity
for adventitious
M the form,
File Application.
If serum or additives
medium,
to CBER,
data directly
of serum
should be determined
responses
be made to reduce
of production
amount
allergic
serum
cell cultures
or additives
and shall not exceed
in human
levels
required
as much as possible.
in the final p~duct
1:1 ,000,000(21
CFR
610.15(b)).
If porcine trypsin
adventitious
113.53).
is used in passaging
including
agents,
Pursuant
porcine
to 21 CFR 207.31,
products
are requested
to provide
source(s)
and control
of any bovine-
(see attachment
#1)-
cells,
it should
parvovirus
(9 CFR 113.51
manufacturers
information
be free from
of biological
regarding
or ovine-derived
the
material(s)
and
14
Penicillin
or other beta Iactem antibiotics
production
cell cultures.
Minimal
or inducing agents may be acceptable
However,
the presence
of any antibiotic
in
of other
concerltrations
antibiotics
product
not be present
should
[21 CFR 61 O.15(C)].
c)r inducing
agent in the
is discouraged.
B. Management
Lot-to-lot
of Cell Cultures
for adventitious
biological
agents
and unprocessed
Appropriate
testing
and processed
approaches
and
is part of the quality
It includes
product.
product
of the
characterization
on the nature of the propagation
cell cultures
fluids.
contrc~l of cell substrate
system used.
depend
Ceil substrates
as monolayer
bioreactors,
and may be held on a short term, long term, or even on a
p{oduct
indefinite
is obtained
or from multiple
When short-term
basis.
either from a single
In some ~:ses
harvests.
may need to be performed
bulk
in suspension
cultures,
are
propagated
potentially
cultures,
monitoring
of the
control
c~f production
cell culture
to quality
routine
cultures
harvest
or in
are used,
of cell culture
the quality
control
the
fluid
testing
on each harvest before pooling into the
lot.
If the product
is an infectious
or more of the cell cultures
viruses.
Nonreplicating
virus,
it will usually
used for routine
viruses,
testing
used as vectors
replicate
for adventitious
for gene therapy,
may be tested in the usual ce[[ culture tests for the presence
adventitious
agents.
In such cases a proportion
in one
of the vessels
of
15
containing
the cell substrate
prepared
for production
about 10% of the vessels) should be helcl as control
uninoculated
adventitious
identity
control
agents.
test, which
neutralized
cell cultures
and fluids
(This should
not % confused
is typically
and inoculated
When long term cultures
into bulk lots at intervals.
should be performed
separated
The management
control
testing
Criteria
testing.
established
Testing
be designed
for termination
the unprocessed
The unprocessed
that constitutes
quality
may be pooled
control
pooled
testing
on cells
into the specific
for the purposes
to optimize
of quality
sensitivity
of long-term
bulk.
cultures
of the
should
be
a homogeneous
agents
be performed
is generally
mixture
performed
toxic in test cell cultures,
processing
is a concentrated,
for mixing
for manufacture
processing
unless
whereas
of cell cuiture
that testing
prior to further
by initial partial
prepared
harvests
It is important
or other procedures,
bulk product
sterility
bulk is the pooled
lot of product.
clarification
and fungal
on
bulk lot, the final bulk lot and the final product.
unique
suspension
is
and followed.
for bacterial
sensitive
harvest
of cell substrates
should
the virus
on each bulk lot, and, if possible,
from the production
for
cell culture.)
hawests
cases
The
with the product
in which
into a susceptible
In these
cultures.
are tested
a procedure
are used, multiple
(commonly,
such
(e.g., unprocessed
bulk
purified
product
with
excipients
into a
for adventitious
such testing
filtered
fluids
as filtration,
is made
more
bulk may be
may not).
Final
in a homogeneous
and filling
into final
16
The final bulk product
containers.
release
tests
which
often
include
Final product
to be sterile.
is subjected
sterility
should
to a variety
testing
be tested
of lot
if it is intended
for sterility
and
endotoxin.
Routine
testing
adventitious
production
viruses
and in vitro and in vivo
should be performed
cells and unprocessed
to produce
testing
for mycoplasmas
bulk fluids.
a virus that is routinely
on a lot-to-lot
present
after purification
product,
as is the case in viral vectors
of production
The presence
of nucleic
has been discussed
Organization
concern
content
as a theoretical
group
was negligible
unless
‘for gene
identity
risk.
recommended
DNA (4).
products
and lot release
produced
limits
purity that can be achieved
reasonably
unique
concern.
tests
that
products
on all praducts
reflect
the quality
In this document
Health
that
this
theoretical
that contained
testing
that
reflect
less
for DNA
be
a level
and consistently.
Other tests which should also be performed
tests that are required
is the
be performed.
in cell lines should
established
the
Lot-to-lot
therapy.
should
Lot-tG-lot
bulk,
from
the virus
A World
or absent in products
of cellular
in biological
performed
for cellular
its absence
acid from cell lines in biological
consultative
than 10C pg/dose
in the unprocessed
may be required,
cells
lot using
If a cell line k known
basis to demonstrate
product
testing
on every
testing foi
on every
(e.g., general
of the specific
the discussion
of testing
lot include
safety)
or
product
of
is limited
to
of
17
those
cell
tests which
lines.
release
Manufacturers
procedures
of product
v.
have specific
relevance
to products
are responsible
that provide
for establishing
assurance
in
lot
of all significant
aspects
quality.
QUALITY CONTROL TESTING
A.
Tests for the Presence of Bacteria
For required test procedures,
B.
Tests for the Presence
Tests for the presence
mycop[asmas
and Fungi
see 21 CFR 610.12.
of Mycoplasma
and non cultivable
of both cultivabll$
Bic~logical products
should be performed.
insect cell lines . should be tested for bc}th mycoplasma
spiroplasma
contamination.
mycoplasma
testing
document.
b
produced
Current
suggested
are described
Acceptable
and
methods
in attachment
tests for spiroplasmas
made in
for
#2 of this
should
be discussed
with CBER.
c.
Tests
for the Presence
1.
Routine
Tests
The cell cultures
production
period
hemadsorbing
at different
for Adventitious
Viruses
should be observed
for viral
viruses.
times,
of Viruses
cytopathic
if multiple
the cultures
at the time of the collection
at the end of the
effects
harvest
should
and tested
pools are prepared
be observed
of each pool.
for
and tested
18
of each unprocessed
At the time of production
proportion
of the
pool should
be inoculated
bulk pool, a
into cell cultures,
eggs, and mice as follows:
a.
An appropriate
monolayer
vulume should be inoculated
cultures
(1) monolayer
tissue
of at least three
cultures
into
cell types:
of the same species
and
type as that used for production;
(2) monolayer
culture;
cultures
bf a human
diploid
cell
and ~~
‘(3) monolayer
cultures
of a monkey
kidney
cell
culture.
The sample to be tested should be diluted
possible.
The cell cultures
least two weeks.
to be capable
cytomegalovirus,
be observed
be tested
period.
should be observ~d
If the production
of supporting
the human
cell culture
I,he growth
diploid
for at least four weeks.
for hemadsorption
as ~ittle as
for at
is known
of human
cell cultures
The cultures
should
should
at the end of the observation
19
b.
Fluids or Iysates Gf t:te test sample being
characterized
for viruses
testing
embryonated
hen eggs, as described
in 21 CFR 630.35,
appropriate.
In some cases, testing
in guinea
Selected
testing
Species-specific
be detected
production
Iymphocytic
in adult and suckling
in animals.
most cases,
rabbits or monkeys
2.
be tested
should
In
mice and
is
pigs,
may also be advisable.
for
adventitious
viruses
viruses
present
in rodent
by mouse, rat, and hamster
tests (MAP, RAP, &
choriomeningitis
lines
may
antibody
HAP).
virus
cell
/r? vivo testing for
(LCM)
including
challenge for non-lethal strains is recommended.
Human
.cell lines may be screened ‘for human virus pathogens
such as Epstein-Barr
virus ~(EBV), cytomegalovirus
(CMV),
and hepatitis B and C (HBV, HCV) using appropriate
vitro techniques.
b
Selection
should take into account
history
01 the patient
derived.
Retrovirus
Use of other cell cultures
characterization
source
of viruses
thf? tissue
from
testing
which
the cell
is discussed
specific
testing
fc)r the
medical
line was
below.
also may be appropriate
for
on the cell type and
of the cell line being characterized
circumstances,
to be screened
source and
of cell banks depending
in
(5).
presence
Under certain
of other
20
transforming
viruses,
3.
viruses,
such as papilloma-,
adeno-
and Herpes
may also be indicated.
Tests
for
Test samples
retroviruses
Retroviruses
should be examined
for the presence
the
techniques:
utilizing
electron
a. transmission
b. reverse
following
transcriptase
microscopy
pellets
obtained
centrifugation
(TEM);
(RT) assays
the presence of magnesium
of
(performed
and manganese)
in
on
from fluid:;’ by high speed
(e.g. 125,000 x g for one hour) at
40C; and
c. infectivity
assays.
amplification
achieved
susceptible
latter
tested
For murine
retroviruses,
of low level contaminants
by co-cultivation
of cells
may be
with
a kighly
cell line, e.g. MUS dunni cells ~6). The
cells are susceptible
murine leukemia
to infection
viruses
(MuLVS) except
Moloney MuLV, in which case another
cell line, e.g. SC-1 (7), should be used.
the co-cultures
should be further
dunni cells and subsequently
by all
susceptible
Fluid from
passaged
assayed
on Mm
for MuLV.
6
21
A variety of other assays may be useful, depending
circumstances.
cell immunofluorescence
(IFA) on the infected
using a broadly
monoclinal
the detection
forming
cells
reactive
of ecotropic,
and amphotropic
(8) for detection
assay for detection
assay
culture
xenotropic,
viruses;
feline
of xenotropic
possible
assays
to increase
by first
any retrovirus
concentrations.
S+ L- assay
viruses;
viruses
(1 O) and mouse S+ L-
a broad range of retroviruses,
non-human
primate
of ecotropic
growth
material
in order
origin
viruses
onto cell
to amplify
at low
test
to support
including
viruses.
of tissue
retroviruses,
for their capacity
using PG4
mink S+ L-
that relay be present
For non-murine
be selected
cell focus-
inoculating’” the test
contaminant
~US dunnj cells
the sensitivity
retroviral
include viable
(e.g. HY95) for
mink
of amphotropic
lines that can support
should
antibody
using D56 (9) cells for detection
It is often
b
01 such assays
Some examples
on the
cell
lines
the growth
of
of human and
(10, 10a).
--
Murine
cell lines or hybrid cell lines containing
component
producing
used
testing
should be considered
infectious
mouse
for monoclinal
manufacturing
inactivation
additional
process
capable
For murine
production,
specific
may be abbreviated.
should
of retroviruses.
concerns
retroviruses.
antibody
and identification
inherently
arise.
a murine
be validated
cell lines
retrovirus
However,
for removal
For murine-human
The manufacturer
of
the
and/or
hybrids,
should
refer to
22
the ‘Points
to Consider
Monoclinal
Antibody
discuss
in the Manufacture
and Testing
Products for Human Use (1987)”
any proposed testing
with the agency
of
and
on a case-by-
case basis.
Probe
hybridization/PCR
amplifica~tion
monoclinal
antibody
detection
information
on the presence
and
may provide
or absence
virus-specific
additional
of specific
contaminants.
D.
Tumorigenicity
Testing
\.\.
As noted
rodents
epithelial
in the introduction,
need not ordinarily
continuous
be tested
cell lines derived
for tumorigenicity.
lines and all lines used for live virus
should, however,
be tested.
In addition,
Human
vaccine
production
in some special cases,
to be used in somatic cell or gene therapy
tumorigenicity
from
cells
may require
testing.
—-
Systems
which may be suitable
for M vivo
1.
nude mice (Nu/Nu) (1 1);
2.
newborn
hamsters,
antithymocyte
serum
testing
include:
mice, or rats immunosuppressed
(ATS)
or globulin
(ATG)
with
(12,13,14);
.
23
3.
thymectomized
reconstituted
In all cases,
animals
should
be inoculated
growing
with antithymocyte
injected
marrow
from
should consist
healthy
mice.
of 107 reference
of serum-free
or intramuscular
route.
medium
At least ten
with test cells vvhich are at or beyond
level and at least-ten
nine out of ten animals
progressively
bone
mice that have been
in a 0.2 ml volume
by the subcutaneous
end-production
group
with
the inoculum
cells or test cells suspended
administered
and irradiated
injected
with reference
with reference
tumor
cells
should
In the case of newborn
tumors.
preparations,
with reference
metastasis
cells which
should
cells.
migh’t include
At least
show
animals
also
the
treated
be evident
among
in the
others,
KB,
HT-1 080, and FL.
In the test systems
using newborn
hamsters,
mice, or rats, the animals
should be injected S.C. or i.m. with 0.1 ml volulmes of potent ATS or ATG on
the day of birth and on days 2, 7, and 14 of life.
one which suppresses
the subsequent
routinely
inoculation
produces
In all test systems,
and frequent
injection.
the immune mechanisms
progressively
the animals
intervals
growing
of the animals
tumors
and
shall be observed
for the formation
Animals
the period of observation
palpable
showing
metastasis.
and palpated
of nodules
at the sites
at regular
of
in two dimensions
nodules which begin to regress
should be sacrificed
and processed
such that
tumor cells on the day of birth
Any nodules formed should be measured
the data recorded.
longer
of reference
A potent ATS or ATG is
for histological
before
the nodules
examination.
and
during
are no
Animals
with
24
progressively
growing
those without
include
before
A necropsy
examination
inoculation
kidneys,
should be observed
nodule formation,
half for 12 weeks
examination.
nodules
be observed
half should
being sacrificed
evidence
Among
for 3 weeks
ancl processed
should be performed
for gross
for 1-2 weeks.
and
for histological
on each animal and will
of tumor
formation
at the site of
and in other organs such as lymph nodes, lungs, brain, spleen,
All tumo -like lesions
and liver.
to be examined
metastasis
In addition,
histologically.
without
evidence
and the site of inoculation
are
since some cell lines may form
of local tumor
growth,
any detectable
regional lymph nodes and the lungs of all animals should be examined
histologically.
\.,.
In addition
to in vivo testing,
the characterization
(15) and growth
sensitive
(12).
assays
These
several
in vitro. test
Both colony
of cell lines.
formation
for tumorigenicity
in vitro systems
These tes;s constitute
or progression
history of a candidate
than tumor
are particularly
cell line.
applicable
in animals
rapid and inexpensive
of abnormal
formation
useful
for
in soft agarose
in nude
mice
to continuous
at low passage
cell
levels.
means of demoflstrating
characteristics
over the passage
If, in the hands of the manufacturer,
these tests are shown to be at least as sensitive
tests, they may in some cases be substituted
V1.
are
in organ culture- (16,17,18) have been shown to be more
lines some of which are non-tumorigenic
stability
systems
VALIDATION OF VIRAL ELIMINATION
as acceptable
for the animal
animal
tests.
the
25
Traditionally,
cell
lines
have been tested
viruses,
used as cell
to assure
qualify
for production,
murine
findings,
with monoclinal
retroviruses,
can be safe and approaches
for contamination
risk
manufacturers
which cause
associated
have
studies
the manufacturing
virus than is present
virus-like
particles
antibodies
evidence
in cell
produced
with
such
and/or
to validate
process
that such
products
both the
retroviruses
and the
In particular,
procedures
that
of viruses
from
the effectiveness
is known
and
in cell lines which
contamination.
removal
lines
As experience
have been develc@ed to minimize
with
and
of intracisterna{
concern.
has accumulated
of the products
to
in an enhanced
particles
theoretical
As
necessary
such as the presence
in the unprocessed
tested for the presence
were used.
have resulted
used manufacturing
inactivation
have performed
produce
production
with adventitious
it has become
of virus-like
are only of remote
has been gained
theoretical
that
These efforts
that certain
type A particles
potential
lines
of the significance
demonstrated
produce
cell
viruses.
understanding
include
steps
the product
and
of the procedures.
to eliminate
significantly
bulk and the purifie~
of virus, there is reasonable
assurance
more
product
is
of freedom
contamination.
Accordingly,
when a cell line used for production
known or suspected
to contain
effectiveness
manufacturing
assist
of contamination
cell lines have been introduced,
even infectious
from
the absence
for biologics
and cell lines free from such contamination
continuous
When
substrates
of the
in qualifying
the MCB.
an infectious
Validation
of risk, but do not of themselves
to evaluation
process
of cell lines carrying
virus,
of a biologic
studies
in eliminating
studies
prove absence
is
to validate
that
virus
the
may
assist
in the quantitation
of risk.
They are relevant
any type of virus
(e.g.,
Epstein-Barr
26
virus,
papilloma
virus)
but risk assessment
includes
consideration
of the
type of virus and the potential
use of the product.
Validation
not a means of demonstrating
that the introduction
of an adventitious
virus
into the cell cultures
Validation
that the manufacturing
accomplished
by demonstration
that adventitious
effectiveness
associated
agents
of virus
removal
validation
purification
is suitable
studies
or suspected
steps
regard
so
to validate
of risk
to carry
infectious
by evaluating
to specifically
is
controlled
to evaluation
may be accomplished
selected
in this
is suitably
Thus,
are only relevant
processing
are
can be acceptable.
that the ptocess
the
remove
The j~roduct is “spiked”
virus from the bulk harvest.
of high titer before testing
A.
process
are not introduced.
of the downstream
inactivate
manufacture
with cell lines that are known
Therefore
viruses.
ability
during
studies
steps in a scaled-down
and/or
with virus
model of the
scheme.
DESIGN
The design
process
of procedures
shouid
include
.
1. Selection
to validate
consideration
of ~
elimination
of virus
of the following
r. te virus or vir~~
by az purification
variab@.
The virus(es)
to be
used may be the virus which is knoyvn or suspected
to contaminate
the cell line or it may be a model virus(es)
because
similarity
to the virus
as availability
contaminant
Iabeled form.
of concern
of material
selected
a,nd practical
considerations
in high titer and ease of assay.
may be added in a iabqled
It may be necessary
of its
(i.e., radioactive)
such
The
or non-
to use more than one virus when,
27
for example, the use of one virus does nc)t provide
for the evaluation
2. ~n
of the adequacy
.
M~g
the purification
accurately
scheme
reflect
sy.@2M. If a
manufacturing
salt, and product should
to full scale
3.
Analysis
desirable
to evaluate
manufacturing
present
manufacturing
.
of &6~!n.
an adequate
testing
and equivalence-
In many cases it is
of ?nore than one
elimination.
Sufficient
before
virus
each critical
evacuation of the effectiveness
its concentration
pH, and concentration
o f~s.
i
to be tested
In some cases simply adding
flow
demonstrated.
.. .
step to virus
it should
Bed size,
process.
types,
model of
process
all be evaluated
the contribution
in the material
scaled-down
is used for this validation
the actual
basis
of the process.
rate, flow rate to bed size ratio, buffer
of protein,
an adequate
cases adding virus to in-process
steps
of each step is obtained.
will
material
be
step so that
high titer virus to unpurified
between
should
bulk and
be sufficient.
In other
will also be needed.
virus titer before and after each. step be!ing tested
The
shou~d be
determined.
4. ~
Ph@cal
val versus
contribution
of each of the purification
determining,
when feasible,
virus inactivation
virus from
steps
.
.
should
The type of
be identified
what porticln of the reduction
and what portion
the product.
ln~
is due to physical
by
is due to
removal
of the
28
5. Kinetics
of Ina ctivatiorl
inactivation
at the critical
In some cases
inactivation
This type of data is particularly
to be a human pathogen
process
is being designed.
6. j%tiw
of~ed
individually
calculated
tested
inactivation/reduction
are depended upon
during
the virus
effective
the total
purification
is known
inactivation
effects
titer,
of each
should
be
virus
procedure.
procedures
When chrdmatographic
Routine procedures
it is critical
that the
as actually
used
for the
regeneration
should be such that the design of the validation
is relevant.
Precg@ions.
The validation
manufacturing
facility
contamination
of the
b.
where
of virus
be determined.
of virus
studies should employ columns
8. -c
a.
should
The combined
for virus elimination,
manufacturing.
of columns
study
of the
of Column&
validation
important
step, on the reduction
in order to establish
7. ~on
step
and a completely
Eff-
the kinetics
-.
testing
is frequently
in order
to prevent
possible
outside
virus
facility.
Care should be taken in preparing
preparation
performed
to avoid aggregation
which
the high titer virus
may enhance
physical
the
29
removal
and decrease
manufacturing
thu:j
distorting
the correlation
with
the
c.
The virus “spike” should be added to the product
volume
actual
inactivation
situation.
so as not to dilute or change
in a small
the characteristics
of the
product.
d.
can
Small differences
substantially
e.
affect
virus
particular
chromatography
the full scale
as these components
are toxic
pi-i, or dialysis
necessary.
the validation
Therefore,
in a parthdar
column
and product
or interference
solutions
or reagents,
should
buffer
reflect
the amount
solution
of
or on a
the conditions
of
process.
.
Buffers
toxicity
remains
media,
clearance.
is time dependent.
Virus inactivation
time a spiked product
f.
in, for examplen buffers,
should be evaluated
in assays
used to determine
may adversely
affect
to the indicator
cells,
of the buffer containing
If the biological
product
dilution,
spiked
itself
run, though
substituting
a similar
could affect
the behavior
omitting
protein
Joes
of the virus
not
in some
titer,
cells.
If the
adjfistrnent’
of the
might be
has an anti-viral
the biological
th~t
virus
for
the virus
the indicator
study may need to be performed
in a “mock”
independently
activity,
without
the product
product
or
have
anti-viral
activity
manufacturing
steps.
30
Many purification
9.
columns
elimination
when analyzing
by a particular
manufacture
B.
use the same or similar
The effects
repetitively.
into account
schemes
at which
of this approach
the data.
process
buffers
should
The effectiveness
or
be taken
of virus
may vary with the stage
in
it is used.
INTERPRETATION:
..
The purpose of a validation
according
to SOP’s,
contaminants,
eliminated,
will reliably
it is important
but that
there
into the purification
the final product.
unpurified
The amount
estimates
microscopy
to examine
level of safety
amount of virus which may be present
it is important
unpurified
that an estimate
bulk is made.
for
in
purification
a pellet
process
When such assays
excess that is appropriate
of ultracentriluged,
should
be able
to be present
depends
intended use of the product.
to eliminate
in the starting
may be very significant,
where
the
a larger
relatively
excess
material.
substantially
material.
on the virus of concern
are
electron
unpurified
For example, for products
individuals,
of the
When ~ossible,
may be made by using transmission
more virus than is thought
infection
built
by the manufacturing
may be done by assays for infectivity.
immunocompromised
elimination
bulk product.
not feasible
The entire
an appropriate
of virus eliminated
amount of virus in the ordinary
this estimate
For virus
capacity ‘Vor virus
to assure
to-the
result.
when done
to show that not only is the virus
To carry out this comparison
\
give a certain
is excess
process
process is compared
, ordinary
study is to show that a process,
The
and the
intended
small
for use in
risk of
in clearance
capability
31
The same increase
may be indicated.
apply in the case of products
The
following
virus
removal
potential
should
in the clearance
capability
intended for use in a healthy
..-”
limitations
of studies
be addressed
when
1.The model virus may not behave
population.
to validate
interpreting
identically
would
of
elimination
study
results.
to the relevant
virus
contaminant.
2. The full-scale
process
may be different
from the scaled
down
process.
..
3. Unpredicted
procedures
similarities
or redundancies
may overestimate
..
4. The summation
steps
with
virus
elimination.
5. The ability
little
virus
of the effects
effect,
of buffer
solutions
or
particularly
of
clearance.
of multiple
may overestimate
steps,
the
true
potential
.
over time of chromatography
used in the purification
scheme
columns
to clear virus
after
and other devices
repeated
may vary.
6. Validation
variation
studies
within
for
should
and between
be duplicated
studies
and the statistical
evaluated.
use
32
7.lt is recommended
virus
inactivation
present
c.
step when
routinely
infectious
in unpurified
scheme
virus
provide
is known
at ieast one
to be
bulk.
STATISTICS
The validation
process
the data to evaluate
valid to support
D.
that a purification
should
include
the use! of a statistical
The study design
the results.
the conclusions
should
analysis
of
be statistically
reached.
REVALIDATION
\...
Whenever
significant
changes
in the -production
made, the effect of that change
and the system revalidated
changes
in production
on virus clearance
as needed.
processes
should
For example,
to cause significant
process
are
be considered
it is not unusual for
changes
by the cell line or removed
amount of virus produced
manufacturing
or purification
in the
by a particular
step.
VII. CONCLUSION
The FDA’s Vaccines
Committee
several
of August
manufacturers
Proceedings
strategies
meeting
and Related
including:
Products
21, 1990, reviewed
to remove
of this meeting
Biological
retrovirus
emphasized
from
Advisory
the approach
their
products.
the value of many
of
33
A.
thorough
starting
characterization/screening
material
in order
of the
to identify
what
cell
substrate
virus
contaminants
are present;
B.
determination
of the human
c.
incorporation
of validated
into the manufacturing
D.
careful
design
and provide
E
virus
interpretable
inactivation
validation
results;
methods
the same manufacturing
of the contaminants;
and
removal
steps
process;
of the virus
use of different
virus
tropism
of virus
process
studies
to avoid
pitfalls
or removal
in
and
inactivation
in order to achieve
maximum
clearance.
\
Validation
studies
should
time during pre-lND
be discussed
meetings
make sure no outstanding
application.
provide
The successful
an approach
with CBER at the earliest
and then again before phase
issues
remain
prior to filing
use of these quality
for producing
safe biological
control
possible
Ill studies
to
a license
elements
products.
should
34
Vlll.
REFEREKES
1.
Continuous
Cell Lines as Substrates
Developments
2.
Continuous
Moorehead,
Petricciani,
4.
Standardization,
Cell Lines: Current
Standardization,
3.
in Biological
for Biological.
Issues.
1989.
Developments
in Biological
1992.
..
P, Earley, EM, Frieiing, K, Jacobs,
J, von Seefried,
P, Litivin, J, Vetter,
A (1979) J. Biol. Standard.,
Report of a WHO Study Group (1987) ~~ceptability
for production
Vol. 70
of biological.
World
7, 397-404.
of cell substrates
t-le:~th Organization
Technical
Report Series 747.
5.
Jacobs, JP, Magrath, Dl, Garrett, AJ, Schild GC (1981) J. Biol.
Standard.
9: 331-342.
6.
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PJ.
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36
Attachment
#1
Letter
to
the
Manufacturers
of
Biological
Products
May 3, 1991
Dear Biologic
Product
Manufacturer:
The Center for Biologics Evaluation and Research (CBER) is seeking
clarification
of the procedures and precautions used in controlling
materials of bovine or ovine origin used in the manufacture of biologic
products intended for administration to humans. This will assist CBER in
evaluating the impact of evolving information--regarding
- infectious
agents
potentially present in materials from bovine or ovine sources (e.g.,
spongiform
encephalopathies).
We are therefore requesting, pursuant to 21 CFR 207.31, that
manufacturers
of biologic products provide information
regarding the
source(s) and control of any bovine- or ovine-derived
material(s) used in
preparing products to be administered to humans for prophylaxis, therapy,
or diagnosis. This request is not only for information
relating to material
that is directly incorporated
into the product, but also for information
on
any materials used in manufacturing (e.g., enzymes, cell culture
components, chromatographic
media, etc.).
Some specific examples of materials that are, or may be, of bovine or
ovine origin include bovine fetal serum, bovine serum albumin, fetuin,
proteolytic enzymes (e.g., protease, trypsin, chymotrypsin,
etc~,
deoxyribonucleases
(this is not intended to be a complete listing). If you
are unsure of the origin of a component usecl in the preparation of your
products, please obtain this information from the supplier.
Please submit the following information regarding each biologic product
that you manufacture under an accepted product license, pending license
application or amendment, or investigational
new drug application (lND)
(This information should not be submitted to your license, license
application or amendment, or IND; see instructions below):
The name and status (licensed,
product.
license pendilng, or IND) of each biologic
37
Page 2 - Biologic
Product
Manufacturer
For each product, a list of the material(s) derived from bovine or
ovine sources used directly in the product or in manufacturing.
If no
material from bovine or ovine sources is used, indicate “none” in response.
The name and address of the supplier(s)
material.
of each bovine-
or ovine-derived
A description of the controls utilized by you and the supplier(s) of bovineor ovine-derived material(s) to assure and document the health and
country of origin of the animals used in production of these materials.
A description of the testing performed on each lot of bovine- or ovinederived material, including the acceptance criteria used. Indicate if the
testing is performed by you or the supplier. If performed by the supplier,
indicate if you receive detailed test results or summary information.
We request
date to:
that you submit this information
Gerald V. Quinnan, Jr., M.D.
Acting
Director
.
Center for Biologics Evaluation and Research
Division of Biostatistics
and Epidemiology
Attention: HFB-250,
Building 29-BSE
8800 Rockville Pike
Bethesda, MD 20892
Sincerely
yours,
Gerald V. Quinnan, Jr., M.D.
Acting
Director
Center for Biologics
Evaluation and Research
within
60 days of the above
38
Attach ment#2
RECOMMENDED
CONTAMINATION
PROCEDURES FOR DETECTION OF MYCOPLASMA
IN BIOLOGICAL
PRODUCTS PRODUCED IN CELL
SUBSTRATES
Each licensed biological product produced in cell substrates
(e.g., viral
vaccines,
monoclinal
antibodies,
immunological
modulators,
interferon
and other cytokines, erythropoietin,
growth factors, and similar products)
For.
must be tested to ensure the absence of mycoplasmal contamination.
most such products, testing should be performed on the virus seed and/or
master cell banks, cell substrate and a representative
portion (not more
than 10 percent) of-each working. celL stock used for manufacture of the
product. Each lot of product harvest concentrate should be tested prior to
and inactivation,
although
testing
at
clarification,
filtration,
purification,
this stage of the manufacturing process may not be appropriate for all
products. Prior to testing, the product harvest concentrate
sample should
generally be stored between 2 and 8° C for 24~hours or less or at -60° C or
lower for 24 hours or more.
As specified in 21 CFR 610.30, mycoplasmal contamination
testing must
be performed by both the agar and broth meciia procedure and the indicator
cell culture procedure or by a procedure demonstrated to be comparable.
The procedural steps recommended for performing both of these
procedures are provided below.
The Center for Biologics Evaluation- and Research
regarding any or all aspects of these procedures.
will provide
guidance
A. A(3AR AND BROTH MEDIA PROCEDURE
(1) Each lot of agar and broth medium should be free of antibiotics except
for penicillin, and each lot of medium should be examined for mycoplasmal
growth-promoting
properties. To demonstrate
the capability of the media
to detect known mycoplasma contaminants,
use the mycoplasmal
cultures
specified below in (3)(i) as positive controls.
(2)(i) Inoculate no less than 0.2 milliliter (ml) of the product harvest
concentr~fe sample in evenly distributed amounts over the surface of 2 or
more agar plates of 1 medium formulation.
39
(ii) Inoculate no less than 10 ml of the product harvest concentrate
sample into a flask containing 50 ml of broth medium which is incubated
at 36+ 1° C.
(iii) Test 0.2 ml of the broth culture on the 3rd, 7th, and 14th days of
incubation by subculture onto 2 or more agar plates of the same medium
formulation as that used above in (i).
(iv) Incubate 2 of the initial isolation plates amd 2 each of the three
subculture plates in a 5 to 10 percent carbon dioxide in nitrogen and/or
hydrogen atmosphere containing less than 0.5 percent oxygen during the
test incubation
period.
(v) Incubate all culture agar plates for no less than 14 days at 36 + 10 C
and observe them microscopically
at 10 time magnification
(1 OOX) or
greater for growth of mycoplasmal colonies.
(3)(i) Include in each test at least 2 known nnycoplasma species or strains
as positive controls, 1 of which should be a dextrose fermenter (i.e., M.
pneumonia
strain. FH or equivalent species or strains) and 1 of which
should be an arginine hydroiyzer (i.e., M. orale strain CH19299 or
equivalent species or strains). Positive contrc]l cultures should be not
more than 15 passages from isolation and should be used in a standard
inoculum of 100 colony forming units (CFU) or 100 color-changing
units
(CCU) or less.
(ii) Include
*
uninoculated
(4) Interpret
specification
agar medium
as a negative
the results of the procedure
detailed below in (C)(1-4).
according
control.
to the
=
—.
B. INDICATOR CELL CULTURE PROCEDURE
(1) Using a Vero cell culture substrate, pretest the procedure by using the
mycoplasma! cultures specified below in (3)(i) as positive controls to
demonstrate
the capability of the cell substrate to detect known
fastidious
mycoplasmal
contaminants.
An equivalent
indicator cell
at least equal
substrate may be acceptable if data demonstrate
sensitivity
for
the
detection
of
known
mycoplasrnal
contaminants.
(2)(i) Inoculate
no less than 1 ml of the procluct harvest concentrate
samples
to 2 or more indicator
cell cultures
grown on cover slips in
dishes
or equivalent
containers.
-
40
(ii) Incubate the cell cultures for 3 to 5 days at 36 + 10 C in a 5 percent
carbon dioxide atmosphere. Examine the cell cultures for the presence of
mycoplasmas by epifluorescence
microscopy using a DNA-binding
fluorochrome,
such as bisbenzimidazole
or an equivalent stain.
(3)(i) include in each test 2 known mycoplasma species or strains as
positive controls (i.e., M. hyorhinis strain DBS 1050, M. orale strain
CHI 9299, or equivalent species and strains), using an inoculum of 100 CFU
or 100 CCU or less.
(ii) Include
as a - negative
control
a non-infected
(4) Interpret the results of the procedure
detailed below in (C)(i) -(iv).
indicator
according
cell
culture.
to the specifications
C. INTERPRETATION OF RESULTS
For the agar and broth media procedure, c~mpare the appearance of the
media inoculated with the product to that of the positive and negative
controls.
(1)
(2) For the indicator ceil culture procedure, using 600 times
magnifications
(600x) or greater, compare the microscopic
appearance
the cultures inoculated with the product to that of the positive and
negative cell controls.
\
of
(3)
Marked cytopathic effects or nuclear chromatin fragmentation
caused
by virus infection that affect the interpretation
of the results can be
minimized by using a specific neutralizing viral antiserum or a
The antisera should also-- be aaded to
nonpermissive cell culture substrate.
the positive and negative controls.
(4)
The product
is considered
satisfactory
for manufacture
if both the
agar and/or broth media procedure
and the indicator
cell culture procedure
show no evidence
of mycoplasmal
contamination
(i.e., growth)
and thus
resemble
the negative
control(s)
for each prc~cedure.
(5)
If mycoplasmas
are recovered,
sp,ecie.s may be useful in determining
contamination.
confirmatory
the probable
testing
source
to establish
of
the