-

Interferon Test Procedures: to in the and Testing of Interferon

Intended for Use .

Of of Center for and

-

Contents

I . Introduction .

3-11I I . Process

A. of Cell Substrate

1 . Source: Leukocytes 2. Source : Cells

Source: Cell 4. Cells Used to Produce Products

B. and

1. Inducers 2.

of Manufacture

I I I . Bulk Lot Tests

IV. Final Container Tests 13-17

A . of Lot

B. y

C. General Safety Test

0. Potency of

.1. Cell 2. Viruse 3. Reference Standards 4. of Confidence Intervals for Test Results.

-

Introduction

a Interferon Test Procedures

1980, February 1, which was to represent areas

of general agreement of participants at a workshop held at the NIH on October

29, 1979. Areas of discussion focused on safety, purity, potency, and tests

of clinical correlates. The resulting suggestions and

(IFN) materials were Intended to facilitate progress in

the IFN products for use. It was

that new the manufacture of IFN nould require re-evaluation of

those suggestions.

This, fact, has been the case. The application of new technologies has

resulted better defined products higher activities;

preparations of all IFN types are becoming more readily available for

clinical trials; are resulting the

of data which can now be subjected to further analysis. It has

become apparent that the Interferon lest Procedures are no longer .

For example, were made for certain bulk and final

container tests using reduced volunes. This was considered reasonable since

there was a desire to conserve material needed for clinical investigation. It .

was not Intended, however, that these efforts to conserve material would be

valid or appropriate as lot sizes increased and more

available. However, in those where the of

IFN is we will consider reduced on a

case-by-case basis.

1

believe it is to prevrous suggestrons the of

these new data and to explore further were, due to lack

of experience, to address. the the

discussed below should be interpreted as what the of

(08) generally expects manufacturers of to drug

t . These points are not and of them may

be to Therefore,- the will revlen the adequacy .

of of on a

Many aspects of and of the facility are not

here. Such specific details and the related concerns of interested manufac

turers should more properly be discussed wrth the 08

and should be included in and product In

general, the Current Good and the

pertinent to and control 210,

211 and 600 et seq.) are applicable.

We hope that guidance prove useful to those interested organlung

a t h e

II. Process

A. of Cell Substrate for

The of of cell substrates to assure

that well lots of can be produced

a provide assurance of product

and safety. The type of needed, therefore, depends on the

nature of the used.

2 . .

1. Leukocytes Coats)

Procedures for and of source leukocytes

to donor methods for blood

storage, and

for the Collection of Leukocytes for further

were January 1981, and of

are on request (Dockets Management

Branch, WA-305, FDA, Room 4-62, 5600 fishers Lane,

20857).

2. Source: Human Cells

Human cell used for the of

should have a nell-defrned passage and be demon

strated to be stable and (Report of Hoc Committee on

Control of Cell

397-404). A cell bank should be established

alrquots are used to production cultures. T h e

cell bank must be to be free from agents e.g.,

bacteria, mycoplasna (see and viruses.

the cell banks should be shown to be non-tunorigenrc in

a model. Also, a of the cell cultures

used for the production of each lot of should be retained as

control cultures and tested for and the absence of

agents.

3

-

I

3 . Source: Continuous Cell

from on cell lines and products

them is needed to resolve

to use of such cell such is available,

much be learned continuous cell substrates

for adequacy, and safety for use. These tests

should toward that will-serve

as a for consensus on the safety

and of cell culture derived biological products.

A cell bank should be and tested for

agents as described A., 2. may be

obtained from the cell bank of the cell line

f o r Karyotype and of the cell

bank should also be and these tests repeated

during production.

Cells Used to Produce Products

The biologic regulations in 21 CFR 610.18 prescribe

for and cultures of

or yeast. cell should be described and

characterized as in II., A., 3. In the following . information would provide a data base for future IFN

and may in solving potentral problems, e.g., an

IFN preparation a or insertion the

nucleotide sequence:

-

-

i a: the sequence of the cloned IFN gene(s)

present the seed stocks used for

b. the of the vector, e.g., enzyme

markers, control of copy

etc . ;

and

a n d

should be

1 . Inducers

a. A of the and/or nature of the

, e.g., structure, b y

products,

b. Adverse of the e.g.,

.

c. A of the procedures used to remove and assay for

residual Inducers.

d. for levels of the

products.

S

2.

a. that the procedure for

IFN adequately removes unwanted from

the final product. confirmatory testing

should demonstrate that added are

removed from the product by the

t procedure.

b. If chromatography used, data relating to the

identity, purrty, and safety of the and the amount of

contaminating the product; and a

of procedures for colunn maintenance,

and storage , as well as

the need for colunn replacement.

c. If IFN derrved from cell results of

tests for residual DNA. the absence of defined tests for

measuring residual biologically active DNA,

to of DNA by the method to

the extent that current technology allows detection

mately 10 pg DNA by It should

noted that tests for DNA assays for,

the presence of oncogenes are evaluated.

6

74

I

/

d. Information to the and of

virus when used as an during the

process.

e. 21 CFR 610.15(b) and requirements for the

of foreign proteins The

of than and their

concentrations the product(s) be evaluated on a

case by case

f . IFN used clinical studies should be as pure

as possible. In general, the specific activities of avail

able partially and HuIFN-beta are

protein and that of are

protein.

In some cases IFN preparations higher

are desirable depending on the of the cell

. and the study design. In each case the

produced should a consistent

activity and, where should be .

7

I

- *

The of an IFN should

on i ts

at 100,000 xg for 2 hours); protease susceptlblllty,

loss of antiviral activity follonlng treatment;

stability; on SDS PAGE (protein and

antiviral activity pattern); of its

monocloual or polyclonal

on hrgh perform&e chromatography (HPLC)

contamination other biological e.g.,

lymphokines I, I I), endogeneous pyrogens ,

proteases; of as a of tune

and temperature; sequence; post trans

lational modification; and state of

Altered forms, they cannot be removed, should be further

characterized when feasible, with regard to molecular

antiviral specific activity, and

IFN products DNA technology are

likely to contain which are not found natural

IFN Therefore, in to

described comments, the procedures should be

considered when product purity for each lot of

recombinant DNAder lved ,

(1) odwn dodecyl sulfate

(SDS-PAGE). SDS PACE could be used to assess

product purity and estimate the apparent molecular

weight of the or the prepara

tion. The gels should be the presence and

absence of reducing agents and molecular weight

standards. Staining both blue

and silver or two

gel can be applied to

evaluate product molecular heterogeneity and the

presence of some components.

the preparation should be by

assay or western blotting. In general, of the

protein should be the expected IFN

IFN aggregates or degradation products should be

and removed found

from the parent molecule. If biologically identical to

the parent molecule, they should be present

amounts in each preparation.

(2) Specific activity. The IFN be to a

constant and consistent In general,

the of recombinant have been in

the order of reference per mg NIH or

other accepted IFN reference

standards should be used.

9

-

-

( 3 ) A c i d T h e

should be determined and be consistent the gene

sequence and/or for the native

IFN. It should display the absence of

or any other components of bacteria or yeast.

( 4 ) - P a r t i a l S e q u e n c e slg.

analysis (for example, and

carboxy-terminal sequence analysis could serve as

important criteria of purity for R-DNA produced proteins

or The sequence data presented in tabular

form should include both the total yield and the

yield for each amino acid at each cycle.

Unexpected heterogeneity the terminus and

terminus due to proteolytic degradation of

proteins produced by R-DNA technology have been revealed

by such sequence analysis.

Performance Liquid Chromatography. method can

be used to assess protein purity. Greater than or equal

to of the protein should elute as a single peak

reverse phase chromatography each of two

solvent systems.

( 6) Mapping. mapping a useful criterion

for comparing a recombinant IFN to its native counter

part IFN or retention samples. In serves

to the IFN preparation. It is an important

10

I I

1

-

=

_

*,

method for the assessment of correct bond

formation. can be

proteases with high performance liquid

chromatography or electrophoresis.

All of the above procedures 1. need not be

conducted for each lot; however, whenever there is a

change in protocol in preparative material used,

e.g., when a new lot of material mtroduced, the

procedure should be re-evaluated for

adequacy to remove

Consistency of Manufacture

products derived from cells are to

abilities inherent complex systems. Product .

tency should, therefore, be by close to

tency of procedures as well as analysis of the

product. Inherent In thisconcept is the need tounderstand what

variables are involved and how they may be affected

manufacture. For example, of IFN are to be

present in products derived from eukaryotic cells may be present

from bacteria. Other biologically-active substances

with important clinical effects such as or . endotoxin may also be present. Cellular enzymes may be present

affect the physical state of IFN the final product, such as

. .

-

by of of Manufacturers should

rigorous quality control procedures to detect and reduce .

Manufacturers should the methods used for production and

of their lndlvldual products to judge

are relevant to product and

should be-evaluated.

Bulk Lot Tests

for the purposes of the suggestrons, the term bulk

as the contents of a single from or

harvests to further I f a

nary bulk prepared and into several separate containers

etc.) final cohtalners are filled, the contents of each of

those , etc.) to be a bulk for

purposes and each should bear a lot nunber.

Tests for mycoplasma should be performed on bulk for . .

products fran or contrnwus cell

see at tachnent) . Alternately , tests for mycoplasma may be performed at any

other point the process provrded the adequately

represents the material a bulk lot and represents a point beyond

which mycoplasna not may to

propose alternate tests for mycoplasma.

I

-

- ---

-

. /

IFN from cells in culture should be tested for

sterility prior to f u r t h e r bulk

prescribed in 21 CFR 610.12.

If a protein added to an IFN the activity

of the bulk lot prior to the addition of the exogenous protein should be

determined. .

IV. Final Container Tests

The of and testing procedures for final containers are as follows:

A. o f l o t : that group of final containers,

identical al l respects, have been filled from a single bulk

lot container without any changes that affect the integrity

of the filling assembly. For testing purposes, a AS

subdivided among more than one operation, each such

will be to be a different final container lot

and each should bear a distinguishing lot nunber.

Pyrogeniclty: the importance of pyrogenicity testing of the

container material has not yet been Many

of purified both native and recombinant, very

low endotoxin levels, as determined by the limulus

1 3

(

--

test, and yet pyrogenlc responses and

is at present whether to

or to a presently undetected,

or factors are involved. therefore recommend that

both the rabbit pyrogen LAL tests be performed on each lot.

tests must be performed a dose of

body to that to be the

dose 21 610.13. not less than 1 and

not greater than 10 ml/kg should be used the rabbit pyrogen test.

If samples are to be sterile should be

used. If a rabbit test observed with dose,

recommended that 0.5 solutions of the final container

material be tested order to level of If N y

which negative results. A test, however,

not of itself be a for rejectron; the

the of pyrogenlc material on a case-by-case .

should be out as recommended by the

lysate manufacturer and the test should controls designed to

show that the being tested does not the

of the test. The patient the degree of

of the and the route of its administration should

all be in the assessment of the of any

lot of IFN (Docket BIOLOGICAL, AND ANIMAL DRUGS

AND MEDICAL DEVICES; AVAILABILITY OF DRAFT FOR USE OF

LYSATE

C. The general safety test (21 CFR performed to . .detect extraneous to all

1 4 . .

-- - -- - a y

-

may be made for prepara-

These would only be

Potency o f virus interference assays are the proce

dures most commonly used to measure potency of

of are also reflected other laboratory procedures

which measure ef fects, but

methods for performing these assays are not generally

available. Assays which measure IFN content such as protein

and radio- and have also been

some lab but the extent to which results

these tests correla e with needs to be

on an d u a l Therefore, potency of

should to be ted virus interference assays. most

method for erforming these tes ts undergo

as more becomes available. At present, the

f o l l o w i n g should be taken account :

1. Cell lines: a variety of cell lines are sensitive to IFN effects

when challenged with viruses; and cell lines of non-hunan origin

may be more to effects of some than cell

1 Unless there is evidence that clinical effects of a

IFN preparation are better cor re la ted

a non-hunan cel l then the use of cell lines more

desirable. In test:.ng for which reference reagents exist,

cell lines should selected are to the refer

ence at the potency. In the case

15

http:test:.ng

---

of gamma the same may apply when a reference

becomes In the absence of availability of a standard

reference, should be tested on a

characterized cell line and compared to a working reference

standard.

2. : vesicular virus,

are suitable for of

IFN potency. however, whichever virus used,

of the amount of virus used extent to

variability of virus affects variability of the assay

results should be and should be the for estab

lishing of acceptability of tests. A

of the virus used should be performed each to

of results.

3 . Reference standards: laboratory working standards should be

established. For alpha (leukocyte, cell, or

recombinant DNA-derived) and beta (fibroblast and recombinant

laboratory standards should be

characterized comparison to generally reference

standards as those by the Until a gamma

reference reagent becomes generally available, each laboratory

should establish an internal standard for assay of potency.

The of results for standards should be

evaluated and of acceptability of test results

16

Potency results for under test should be ----

by to results for the standard

the individual tests.

4 . o f c o n f i d e n c e intervals for teat results: well

potency teat procedures should be evaluated for

t . The nunber of required tests and

the-nunber of repeat teats to grve adequate of

should be established. Confidence intervals should

be determined for each final potency result and should reflect

reasonable consistency of the assay.

In t o t e s t i n g for p o t e n c y virus

manufacturers should consider the relevance of tests of

other t o o f ac t ion in

studies. Any testing would

of effects encouraged. I m m u n o a s s a y a o f I F N

potency offer the advantages of improved of

performance, and However, the suit

such assays for routine potency must be

established individually in any laboratory to d o by

that potency results through

correlate blologlcal activity under a variety

0 f The of needed to

should be through

the 08.

1 7

- =

Test for Mycoplasma in Interferon Bulk Material (Lymphoblastoid, Fibroblast, or Other Cell Line Produced Material)

Prior to clarification (or filtration), each lot shall be tested for

the presence of mycoplasmas, as follows: samples of the bulk lot

shall be stored before use either: between and for no

longer than 24 hours; or at or lower if stored for longer

than 24 hours.- The bulk lot tested for the presence of

mycoplasma by using an agar and broth procedure. The media shall be

such as have been shown to be capable of detecting known

and each test shall include control cultures of at least two known

strains of one of which must be pnuemoniae. Each lot

of agar and broth media used shall be examined for growth-promoting

properties and shown to be capable of detecting mycoplasma

contamination as.described below.

No less than 0.4 ml of the bulk lot shall be inoculated, in evenly

distributed amounts, over the surface of four or more plates of agar

media; and no less than ml of the bulk lot shall be inoculated into

25 ml of broth medium. After 3 and 14 days of incubation, 0.4 ml of

the broth cultures shall be tested by subculture onto four or more

agar plates. One-half of the initial isolation plates and one-half of

the subculture plates shall be incubated aerobically in containers

designed to prevent dessication, and the remaining culture plates

incubated in a 5 to 10% carbon dioxide in nitrogen atmosphere. Agar

and broth cultures shall be incubated at 35

18

-

. , . .

All inoculated plates shall be for less than 14 days and observed

for growth of colonies microscopically at no less than 300 tunes

If the (methylene blue-azure dye) or an equivalent

procedure is used to plates for colony growth, at least one

square cm plug of the agar must be The presence of the Mycoplasma

shall be by comparison of the growth the test samples

with that of control cultures, to typical coloni-al and

microscopic . The bulk lot satisfactory none of the tests on

the samples show evidence of the presence of Mycoplasma.

19'

Structure BookmarksFigureFigureInterferon Test Procedures: to in the and Testing of Interferon Intended for Use . FigureFigureFigureFigureFigureFigure

FigureOf of Center for and FigureFigureFigureFigureFigure

PNoteFigure

FigureContents Contents

FigureI. Introduction. . Figure

3-11II. Process Figure

A. of Cell Substrate Figure

1. Source: Leukocytes Figure

2. Source : Cells FigureFigure

Source: Cell FigureFigureFigureFigure

4. Cells Used to Produce Products FigureFigure

FigureB. and FigureFigure

PFigure

1. 1. 1. Inducers

2. 2. LBodyFigure

of Manufacture FigureFigure

III. Bulk Lot Tests. Figure

IV. Final Container Tests. 13-17 A. of Lot FigureFigureFigure

B. y Figure

C. General Safety Test 0.. Potency of ..FigureFigure

1. Cell Figure

2. 2. 2. Viruse Figure

3. 3. Reference Standards

4. 4. of Confidence Intervals for Test Results. Figure

TableTRTHFigure

THFigureFigure

THFigure

TRTHFigure

Introduction TDFigureFigure

TDFigure

TRTHFigure

TDFigureFigure

a TDFigure

TDFigure

Interferon Test Procedures

1980, February 1, which was to represent areas of general agreement of participants at a workshop held at the NIH on October 29, 1979. Areas of discussion focused on safety, purity, potency, and tests of clinical correlates. The resulting suggestions and (IFN) materials were Intended to facilitate progress in FigureFigureFigureFigureFigureFigureFigureFigureFigure

FigureFigurethe IFN products for use. It was that new the manufacture of IFN nould require re-evaluation of those suggestions. FigureFigureFigureFigureFigureFigure

This, fact, has been the case. The application of new technologies has resulted better defined products higher activities; FigureFigureFigureFigureFigure

preparations of all IFN types are becoming more readily available for clinical trials; are resulting the FigureFigureFigureFigureFigureFigure

of data which can now be subjected to further analysis. It has become apparent that the Interferon lest Procedures are no longer . For example, were made for certain bulk and final container tests using reduced volunes. This was considered reasonable since FigureFigureFigureFigure

there was a desire to conserve material needed for clinical investigation. It .was not Intended, however, that these efforts to conserve material would be valid or appropriate as lot sizes increased and more available. However, in those where the of IFN is we will consider reduced on a case-by-case basis. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

1 PFigureFigure

believe it is to prevrous suggestrons the of these new data and to explore further were, due to lack FigureFigureFigureFigureFigureFigureFigureFigureFigure

of experience, to address. the the FigureFigureFigureFigureFigure

discussed below should be interpreted as what the of (08) generally expects manufacturers of to drug FigureFigureFigureFigureFigureFigureFigure

t . These points are not and of them may FigureFigureFigureFigure

be to Therefore,- the will revlen the adequacy FigureFigureFigureFigure

.. of of on a FigureFigureFigureFigureFigure

Many aspects of and of the facility are not here. Such specific details and the related concerns of interested manufacturers should more properly be discussed wrth the 08 and should be included in and product In general, the Current Good and the pertinent to and control 210, 211 and 600 et seq.) are applicable. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

We hope that guidance prove useful to those interested organlung a the FigureFigureFigureFigureFigureFigure

II. Process Figure

A. of Cell Substrate for FigureFigureFigure

PFigure

The of of cell substrates to assure FigureFigureFigure

that well lots of can be produced FigureFigureFigureFigure

a provide assurance of product and safety. The type of needed, therefore, depends on the nature of the used. FigureFigureFigureFigureFigureFigure

2 . FigureFigure

. PFigure

PFigure

Figure1. Leukocytes Coats) FigureFigure

Procedures for and of source leukocytes. to donor methods for blood storage, and were January 1981, and of. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigurefor the Collection of Leukocytes for further. FigureFigureFigure

FigureStyleSpanFigure

FigureFigure

are on request (Dockets Management. Branch, WA-305, FDA, Room 4-62, 5600 fishers Lane, 20857).. FigureFigureFigureFigureFigure

2. Source: Human Cells Figure

PFigure

Human cell used for the of should have a nell-defrned passage and be demonstrated to be stable and (Report of Hoc Committee on Control of Cell 397-404). A cell bank should be established alrquots are used to production cultures. The cell bank must be to be free from agents e.g., bacteria, mycoplasna (see and viruses. the cell banks should be shown to be non-tunorigenrc in a model. Also, a of the cell cultures used for the production of each lot of should be retained as control cultures and tested for and tFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

PFigure

PNoteFigure

Figure3. Figure3. Source: Continuous Cell Figure

Figurefrom on cell lines and products FigureFigureFigure

them is needed to resolve FigureFigureFigureFigureFigure

to use of such cell such is available, FigureFigureFigure

much be learned continuous cell substrates FigureFigureFigureFigure

for adequacy, and safety for use. These tests FigureFigure

should toward that will-serve FigureFigureFigureFigure

as a for consensus on the safety FigureFigureFigure

and of cell culture derived biological products. Figure

A cell bank should be and tested for FigureFigure

agents as described A., 2. may be FigureFigureFigureFigure

obtained from the cell bank of the cell line FigureFigure

for Karyotype and of the cell FigureFigureFigure

bank should also be and these tests repeated Figure

during production. Figure

Cells Used to Produce Products FigureFigureFigure

The biologic regulations in 21 CFR 610.18 prescribe FigureFigure

PFigure

Figurefor and cultures of. or yeast. cell should be described and. characterized as in II., A., 3. In the following. FigureFigureFigureFigureFigureFigureFigureFigure

. information would provide a data base for future IFN and may in solving potentral problems, e.g., an IFN preparation a or insertion the. FigureFigureFigureFigureFigureFigureFigure

FigureFigureFigureFigure

FigureFigurenucleotide sequence: FigurePFigureFigureFigure

i. Figure

a: the sequence of the cloned IFN gene(s) FigureFigure

present the seed stocks used for FigureFigureFigure

b.. the of the vector, e.g., enzyme markers, control of copy etc.; FigureFigureFigureFigureFigureFigure

PFigureFigure

PFigure

and FigureFigureFigure

and should be FigureFigureFigureFigureFigureFigureFigureFigure

1.. Inducers a. A. of the and/or nature of the , e.g., structure, byproducts, FigureFigureFigureFigureFigureFigureFigureFigure

b.. Adverse of the e.g., . FigureFigureFigureFigureFigureFigure

Figurec. .c. .c. .A of the procedures used to remove and assay for residual Inducers. Figure

d.. d.. for levels of the FigureFigureFigureFigureFigure

products. S FigureFigure2.. Figure

Figurea. .that the procedure for. IFN adequately removes unwanted from. the final product. confirmatory testing. should demonstrate that added are. removed from the product by the t procedure.. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

Figureb.. If chromatography used, data relating to the. identity, purrty, and safety of the and the amount of. contaminating the product; and a of procedures for colunn maintenance, and storage , as well as the need for colunn replacement.. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

c.. If IFN derrved from cell results of. tests for residual DNA. the absence of defined tests for. measuring residual biologically active DNA, to of DNA by the method to. the extent that current technology allows detection mately 10 pg DNA by It should noted that tests for DNA assays for,. the presence of oncogenes are evaluated.. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

6 FigurePFigure

PFigure

PParagraphSpanFigureFigureFigure

ParagraphSpanFigure

d.. d.. d.. Information to the and of virus when used as an during the process. FigureFigureFigureFigureFigure

e.e. 21 CFR 610.15(b) and. requirements for the of foreign proteins The FigureFigureFigureFigureFigureFigure

PFigure

of than and their FigureFigureFigureFigure

concentrations the product(s) be evaluated on a case by case FigureFigureFigure

f. IFN used clinical studies should be as pure FigureFigure

as possible. In general, the specific activities of available partially and HuIFN-beta are protein and that of are protein.. FigureFigureFigureFigureFigureFigureFigure

In some cases IFN preparations higher are desirable depending on the of the cell FigureFigureFigureFigureFigureFigure

.. and the study design. In each case the produced should a consistent activity and, where should be FigureFigureFigureFigureFigureFigureFigure

. .

PFigure

Figure7 7

FigureFigurePFigureFigureFigureFigureFigureFigureFigureFigureFigureStyleSpanFigure

The of an IFN should FigureFigureFigure

on its FigureFigureFigureFigure

at 100,000 xg for 2 hours); protease susceptlblllty, Figure

loss of antiviral activity follonlng treatment; FigureFigure

stability; on SDS PAGE (protein and FigureFigureFigure

antiviral activity pattern); of its monocloual or polyclonal FigureFigureFigureFigureFigure

on hrgh perform&e chromatography (HPLC) FigureFigureFigureFigure

contamination other biological e.g., FigureFigure

lymphokines I, I I), endogeneous pyrogens , Figure

proteases; of as a of tune FigureFigureFigure

and temperature; sequence; post transFigureFigureFigure

lational modification; and state of Altered forms, they cannot be removed, should be further characterized when feasible, with regard to molecular antiviral specific activity, and FigureFigureFigureFigure

IFN products DNA technology are likely to contain which are not found natural IFN Therefore, in to described comments, the procedures should be considered when product purity for each lot of recombinant DNAder lved FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

PFigure

,Figure

PFigure

PFigure

PFigure

FigureFigure (1) FigureFigureodwn dodecyl sulfate FigureFigureFigure

Figure). SDS PACE could be used to assess (SDS-PAGEFigure

product purity and estimate the apparent molecular weight of the or the preparaFigureFigureFigure

tion. The gels should be the presence and FigureFigure

absence of reducing agents and molecular weight Figure

standards. Staining both blue FigureFigureFigure

PFigure

and silver or two FigureFigureFigureFigureFigure

gel can be applied to evaluate product molecular heterogeneity and the presence of some components. the preparation should be by assay or western blotting. In general, of the FigureFigureFigureFigureFigureFigureFigureFigure

protein should be the expected IFN Figure

IFN aggregates or degradation products should be and removed found from the parent molecule. If biologically identical to the parent molecule, they should be present FigureFigureFigureFigureFigureFigureFigure

amounts in each preparation. Figure

(2) . The IFN be to a constant and consistent In general, the of recombinant have been in the order of reference per mg NIH or other accepted IFN reference standards should be used. 9 Specific activityFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

FigureFigureFigure(3) Acid The FigureFigureFigureFigureFigureFigure

should be determined and be consistent the gene sequence and/or for the native IFN. It should display the absence of or any other components of bacteria or yeast. FigureFigureFigureFigureFigureFigureFigureFigureFigure

PFigure

(4) -slg. Partial Sequence Figure

FigureFigureFigure

analysis (for example, and carboxy-terminal sequence analysis could serve as important criteria of purity for R-DNA produced proteins or The sequence data presented in tabular form should include both the total yield and the yield for each amino acid at each cycle. Unexpected heterogeneity the terminus and terminus due to proteolytic degradation of FigureFigureFigureFigureFigureFigureFigureFigure

proteins produced by R-DNA technology have been revealed by such sequence analysis. Figure

. method can FigurePerformance Liquid ChromatographyFigure

Figure

be used to assess protein purity. Greater than or equal to of the protein should elute as a single peak reverse phase chromatography each of two FigureFigureFigure

solvent systems. Figure

Figure( 6) . mapping a useful criterion MappingFigure

FigureFigure

PFigure

for comparing a recombinant IFN to its native counterpart IFN or retention samples. In serves FigureFigure

to the IFN preparation. It is an important 10 FigureFigure

FigureFigureI I FigurePFigureFigureFigure

PFigure

method for the assessment of correct bond formation. can be proteases with high performance liquid chromatography or electrophoresis. FigureFigureFigureFigureFigureFigureFigureFigureFigure

All of the above procedures 1. Figure

need not be conducted for each lot; however, whenever there is a change in protocol in preparative material used, e.g., when a new lot of material mtroduced, the procedure should be re-evaluated for adequacy to remove FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

Consistency of Manufacture Figure

products derived from cells are to abilities inherent complex systems. Product FigureFigureFigureFigureFigureFigureFigure

. tency should, therefore, be by close to tency of procedures as well as analysis of the product. Inherent In thisconcept is the need tounderstand what variables are involved and how they may be affected manufacture. For example, of IFN are to be present in products derived from eukaryotic cells may be present from bacteria. Other biologically-active substances with important clinical effects such as or FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

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. Figure

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for the purposes of the suggestrons, the term bulk as the contents of a single from or FigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

Figureharvests to further If a nary bulk prepared and into several separate containers etc.) final cohtalners are filled, the contents of each of FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

those , etc.) to be a bulk for purposes and each should bear a lot nunber. FigureFigureFigureFigureFigureFigureFigureFigure

Tests for mycoplasma should be performed on bulk for products fran or contrnwus cell see at tachnent) . Alternately , tests for mycoplasma may be performed at any FigureFigure. . FigureFigureFigureFigureFigure

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IFN from cells in culture should be tested for sterility prior to further bulk prescribed in 21 CFR 610.12. FigureFigureFigureFigureFigureFigureFigureFigure

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determined. . FigureIV.. Final Container Tests

The of and testing procedures for final containers are as follows: Figure

A.. of lot: that group of final containers, identical all respects, have been filled from a single bulk lot container without any changes that affect the integrity of the filling assembly. For testing purposes, a AS subdivided among more than one operation, each such will be to be a different final container lot and each should bear a distinguishing lot nunber. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

FigurePyrogeniclty: the importance of pyrogenicity testing of the container material has not yet been Many FigureFigureFigureFigure

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dose 21 610.13. not less than 1 and. FigureFigureFigureFigureFigureFigure

not greater than 10 ml/kg should be used the rabbit pyrogen test. Figure

If samples are to be sterile should be. used. If a rabbit test observed with dose, recommended that 0.5 solutions of the final container. material be tested order to level of If N y. which negative results. A test, however,. not of itself be a for rejectron; the the of pyrogenlc material on a case-by-case .. should be out as recommended by the. lysate manufacturer and the test should controls designed to. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

show that the being tested does not the. of the test. The patient the degree of. FigureFigureFigureFigureFigure

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virus interference assays are the procedures most commonly used to measure potency of of are also reflected other laboratory procedures FigureFigureFigureFigureFigure

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some lab but the extent to which results these tests correla e with needs to be on an dual Therefore, potency of should to be ted virus interference assays. most method for erforming these tests undergo as more becomes available. At present, the FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

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1.. Cell lines: a variety of cell lines are sensitive to IFN effects when challenged with viruses; and cell lines of non-hunan origin may be more to effects of some than cell 1 Unless there is evidence that clinical effects of a IFN preparation are better correlated FigureFigureFigureFigureFigureFigureFigureFigure

a non-hunan cell then the use of cell lines more desirable. In for which reference reagents exist, cell lines should selected are to the reference at the potency. FigureFigureFiguretest:.ng FigureFigureFigureFigureFigureIn the case

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characterized cell line and compared to a working reference. standard.. 2.. 2.. 2.. : vesicular virus, are suitable for of IFN potency. however, whichever virus used, of the amount of virus used extent to variability of virus affects variability of the assay results should be and should be the for establishing of acceptability of tests. A of the virus used should be performed each to of results. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

3.. 3.. Reference standards: laboratory working standards should be established. For alpha (leukocyte, cell, or recombinant DNA-derived) and beta (fibroblast and recombinant laboratory standards should be characterized comparison to generally reference standards as those by the Until a gamma reference reagent becomes generally available, each laboratory should establish an internal standard for assay of potency. FigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

The of results for standards should be evaluated and of acceptability of test results 16 FigureFigureFigureFigure

FigureFigureFigurePotency results for under test should be ----by to results for the standard the individual tests. FigureFigureFigureFigureFigureFigureFigureFigureFigure

4.. of confidence intervals for teat results: well potency teat procedures should be evaluated for t . The nunber of required tests and FigureFigureFigureFigureFigureFigureFigure

the-nunber of repeat teats to grve adequate of. should be established. Confidence intervals should. be determined for each final potency result and should reflect. reasonable consistency of the assay.. FigureFigureFigureFigure

In to testing for potency virus manufacturers should consider the relevance of tests of. other to of action in. studies. Any testing would of effects encouraged. Immunoassaya of IFN. potency offer the advantages of improved of. performance, and However, the suitsuch assays for routine potency must be. established individually in any laboratory to do by. that potency results through correlate blologlcal activity under a variety. 0 f The of needed to should be through FigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigureFigure

the 08. 17 Test for Mycoplasma in Interferon Bulk Material. (Lymphoblastoid, Fibroblast, or Other Cell Line Produced Material). Prior to clarification (or filtration), each lot shall be tested for. the presence of mycoplasmas, as follows: samples of the bulk lot. shall be stored before use either: between and for no. longer than 24 hours; or at or lower if stored for longer. FigureFigureFigureFigureFigure

Figurethan 24 hours.-The bulk lot tested for the presence of. mycoplasma by using an agar and broth procedure. The media shall be. such as have been shown to be capable of detecting known and each test shall include control cultures of at least two known. strains of one of which must be Each lot. of agar and broth media used shall be examined for growth-promoting. properties and shown to be capable of detecting mycoplasma. contamination as.described below.. FigureFigureStyleSpanFigure

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No less than 0.4 ml of the bulk lot shall be inoculated, in evenly. distributed amounts, over the surface of four or more plates of agar. media; and no less than ml of the bulk lot shall be inoculated into. 25 ml of broth medium. After 3 and 14 days of incubation, 0.4 ml of. the broth cultures shall be tested by subculture onto four or more. agar plates. One-half of the initial isolation plates and one-half of. the subculture plates shall be incubated aerobically in containers. designed to prevent dessicatiFigure

incubated in a 5 to 10% carbon dioxide in nitrogen atmosphere. Agar. and broth cultures shall be incubated at 35 FigureFigure

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All inoculated plates shall be for less than 14 days and observed for growth of colonies microscopically at no less than 300 tunes If the (methylene blue-azure dye) or an equivalent procedure is used to plates for colony growth, at least one square cm plug of the agar must be The presence of the Mshall be by comparison of the growth the test samples with that of control cultures, to typical coloni-al and FigureFigureFigureFigureFigureFigureFigureFigureycoplasma FigureFigureFigureFigureFigure

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Figuremicroscopic . The bulk lot satisfactory none of the tests on the samples show evidence of the presence of M. FigureFigureFigureycoplasma

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