GUIDANCE FOR INDUSTRY 

FOR THE SUBMISSION OF CHEMISTRY, MANUFACTURING, AND CONTROLS 
INFORMATION FOR A THERAPEUTIC RECOMBINANT DNA-DERIVED PRODUCT 
OR A MONOCLONAL ANTIBODY PRODUCT FOR IN VIVO USE 
Center for Biologics Evaluation and Research (CBER) 
Center for Drug Evaluation and Research (CDER) 
August 1996  TABLE OF CONTENTS 

I.	  INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.  . . .

II. DRUG SUBSTANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.  . .

A. Description And Characterization . . . . . . . . . . . . . . . . . . . . . . . 2.
 
1. Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 
 
2. Characterization / Proof Of Structure  . . . . . . . . . . . . . . . . . .  2
 
a.	  Physicochemical Characterization of Reference . . . . . . .  2
 
Standard and Qualifying Lots
 
b.	  Biological Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
 
 
B. Manufacturer(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 .  . .

1.	  Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 .  . .

2.	  Floor Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.  .

3.	  Other Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 . 

4.	  Contamination Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.
 
 
C. Method(s) Of Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 .
 
1. Raw Materials And Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 6.
 
2.	  Flow Charts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.  .

   
3.	  Detailed Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 .
 
a.	  Animal Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.  .

b.	  Cellular Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 .
 
i.	  Cell Substrate / Host Cell / Expression . . . . . . . . . . . .  8
 
Vector System
 
i
  A.  Recombinant DNA Products Including . . . . . . . . . . .8
 
RDNA-Derived Monoclonal Antibodies.
 
I.	  Host Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 .  .

II.	  Gene Construct . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
 
 
  III.	  Vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 .  .

IV.	  Final Gene Construct . . . . . . . . . . . . . . . . . . . . . 9.  .

V.  Cloning And Establishment Of The . . . . . . . . . . 9. 
 
Recombinant Cell Lines.
 
B.	  Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . 1. 0.
 
ii. 	 Cell Seed Lot System . . . . . . . . . . . . . . . . . . . . . . .1 .0 . 

A.	  Master Cell Bank (MCB) . . . . . . . . . . . . . . . . . . . . . 1. 0. 
B.	  Working Cell Bank . . . . . . . . . . . . . . . . . . . . . . . . . 1. 1.  .

C.	  End Of Production Cells (EPC) . . . . . . . . . . . . . . . . . . 11

 iii.  Cell Growth And Harvesting . . . . . . . . . . . . . . . . . . . . .1 .2
 
C. Purification And Downstream Processing . . . . . . . . . . . . .13
 
4.	  Batch Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 3. 
D. 	 Process Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 4.
 
1.  In-Process Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 .4
  
2.  Process Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 4.
 
a.	  Validation Studies For The Cell Growth And . . . . . . . . . . .14
 
Harvesting Process. 

b.	  Validation Studies For The Purification Process . . . . . . .  14

 c.  Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 .5 . 

ii
  E.  Reference Standard(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 .5 .  . 
1.  Primary Reference Standard . . . . . . . . . . . . . . . . . . . . . . . . . .1 .5
  
2.  Working Reference Standard (If Used) . . . . . . . . . . . . . . . . . .1 5
 
F.  Specifications / Analytical Methods . . . . . . . . . . . . . . . . . . . . .1 .6
  
1.  Drug Substance Specifications And Tests . . . . . . . . . . . . . . . 16
 
a.  Specifications And Analytical Methods . . . . . . . . . . . . . . . 16
 
b.  Certificates Of Analysis And Analytical Results . . . . . .16
 
2.  Impurities Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 6.  .

G. Container/Closure System . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 6.  .

  
H. Drug Substance Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7
 
III. DRUG PRODUCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 .7 . 

A.  Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 .7 . 

B.  Specifications & Methods For Drug Product Ingredients . . . . .1 7
 
1.  Drug Substances Including All Active Ingredients . . . . . . .1 .7
 
2.  Excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 8.
 
a.  Compendial Excipient(s) . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8 

b.  Non-Compendial Excipient(s) . . . . . . . . . . . . . . . . . . . . . . 1.8
 
C. Manufacturer(s). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 .8 . 

D. Methods Of Manufacture And Packaging . . . . . . . . . . . . . . . . . . .1 8
 
   
E.  Specifications & Test Methods For Drug Product . . . . . . . . . . .  19
 
1.  Sampling Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 9.
 
2.  Specifications & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.9
 
iii
  F.  Container/Closure System . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 9.  .

G. Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 .0 . 

H. Drug Product Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 .0 .  .

IV. INVESTIGATIONAL PRODUCT/FORMULATION  . . . . . . . . . . . . . . . . . . . .20
 
V.  ENVIRONMENTAL ASSESSMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
 
VI.  METHOD VALIDATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 .1 . 

VII.  REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 1.  .

iv
  GUIDANCE FOR INDUSTRY1

 FOR THE SUBMISSION OF
 
CHEMISTRY, MANUFACTURING, AND CONTROLS INFORMATION
 
FOR A THERAPEUTIC RECOMBINANT DNA-DERIVED PRODUCT OR 

A MONOCLONAL ANTIBODY PRODUCT FOR IN VIVO USE
 
I.  INTRODUCTION
 
In the Federal Register of May 14, 1996, the Food and Drug 
Administration published the final rule ?Elimination of the 
Establishment License Application for Specified Biotechnology 
and Specified Synthetic Biological Products.  Under this rule 
manufacturers of therapeutic recombinant DNA-derived products 
and/or monoclonal antibody products for in  vivo use are no 
longer required to submit an Establishment License Application 
and may use the interim FDA Form 3439.  This document provides 
guidance on the content and format of the Chemistry, 
Manufacturing, and Controls (CMC) section of a Biologics 
License Application for therapeutic recombinant DNA-derived 
products and monoclonal antibody products for in  vivo use. 
II. DRUG SUBSTANCE
   
The drug substance is defined as the unformulated active
 
substance which may be subsequently formulated with excipients
 
to produce the drug product.
 
0 0  0
This guidance is an informal communication under 21 CFR 10.90(b)(9) that represents the 
best judgement of employees of the Center for Biologics Evaluation and Research (CBER) and the Center 
for Drug Evaluation and Research (CDER), at this time. This statement does not necessarily represent the 
formal position of CBER or CDER and does not bind or otherwise obligate CBER or CDER to the views 
expressed. For further information about this guidance, contact Neil Goldman, Ph.D., Associate Director 
for Research, Center for Biologics Evaluation and Research, 4401 Rockville Pike, Rockville, MD 20852 
(Phone: 301-827-0375; Fax: 301-827-0440) or Yuan-Yuan Chiu, Ph.D., Supervisory Chemist, Biotechnology 
Subcommittee, Center for Drug Evaluation and Research, 5600 Fishers Lane, Rockville, MD 20857 (Phone: 
301-443-3510; Fax: 301-443-9282).  A. Description and Characterization
 
1. Description
 
A clear description of the drug substance should
 
be provided.  This description may include, but
 
not be limited to, any of the following:
 
chemical structure, primary and subunit
 
structure, molecular weight, molecular formula,
 
established USAN name, antibody class/subclass
 
(if appropriate), etc.
 
2. Characterization / Proof of Structure
 
a.	  Physicochemical Characterization of
 
Reference Standard and Qualifying Lots:
 
A description and the results of all the
 
analytical testing performed on the
 
manufacturers reference standard lot and
 
qualifying lots to characterize the drug
 
substance should be included (See
 
references 7, 8, 10).  Information from
 
specific tests regarding identity, purity,
 
stability and  consistency of manufacture
 
of the drug substance should be provided. 

Examples of analyses for which information
 
may be submitted include, but are not
 
necessarily limited to the following:
 
	  amino acid analysis
 
	  amino acid sequencing, entire
 
sequence or amino- and carboxy
terminal sequences
 
	  peptide mapping
 
	  determination of disulfide
 
linkage
 
	  Sodium Dodecyl Sulfate-

Polyacrylamide Gel
 
Electrophoresis (SDS-PAGE)
 
(reduced and non-reduced)
 
	  isoelectric focusing
 
	  Conventional and High Pressure
 
Liquid Chromatography (HPLC)
 
e.g., reverse-phase, size
 
exclusion, ion-exchange, etc.
 
	  mass spectroscopy
  	  assays to detect product-related
 
proteins including deamidated,
 
oxidized, cleaved, and aggregated
 
forms and other variants e.g.,
 
amino acid substitutions,
 
adducts/derivatives.
 
	  assays to detect residual host
 
proteins, DNA, reagents
 
	  immunochemical analyses
 
	  assays to quantitate bioburden,
 
endotoxin
 
Additional physicochemical characterization
 
may be required for products undergoing
 
post-translational modifications, for
 
example, glycosylation, sulfation,
 
phosphorylation, or formylation. 

Additional physicochemical characterization
 
may also be required for products
 
derivatized with other agents, including
 
other proteins, toxins, drugs,
 
radionuclides, or chemicals.  The
 
information submitted should include the
 
degree of derivatization or conjugation,
 
the amount of unmodified product, removal
 
of free materials (e.g., toxins,
 
radionuclides, linkers, etc.), and the
 
stability of the modified product.
 
All test methods should be fully described
 
and the results provided. The application
 
should also include the actual data such as
 
legible copies of chromatograms,
 
photographs of SDS-PAGE or agarose gel,
 
spectra, etc.
 
b.	  Biological Activity
 
A description and results of all relevant
 
in vivo and in vitro biological testing
 
performed on the manufacturers reference
 
standard lot to show the potency and
 
activity(ies) of the drug substance should
 
be provided.  Results of relevant testing
 
performed on lots other than the reference
 
standard lot, that might have been used in
 
establishing the biological activity of the
  product, should also be included.  (See
 
references 7 - 10, 12)  The description and
 
validation of the bioassays should include
 
the methods and standards used, the inter-

and intra-assay variability, and the
 
acceptable limits of the assay. 
 B. Manufacturer(S)
 
1.  Identification
 
The application should include the name(s),
 
address(es), FDA registration number, and other
 
pertinent organizational information for each
 
manufacturer performing any portion of the
 
manufacture or testing operations for the drug
 
substance.  This may include contractors or
 
company subsidiaries serving as contractors, or
 
other locations/sites owned and operated by the
 
applicant.  A brief description of the
 
operations performed at each location, the
 
responsibilities conferred upon each party by
 
the applicant and a description of how the
 
applicant will ensure that each party fulfills
 
their responsibilities should be submitted. 

2.  Floor Diagram(s)
 
For each manufacturing location, a floor diagram
 
should be included that indicates the general
 
facility(ies) layout.  This diagram need not be
 
a detailed engineering schematic or blueprint,
 
but rather a simple drawing that depicts the
 
relationship of the subject manufacturing areas,
 
suites, or rooms to one another, and should
 
indicate other uses made of adjacent areas that
 
are not the subject of the application.   This
 
diagram should be sufficiently clear such that
 
the reviewer may visualize the flow of the
 
production of the drug substance and would be
 
able to identify areas or room "proximities"
 
that may be of concern for particular
 
operations, e.g. segregation of pre and post
 
viral inactivation material and operations,
 
segregation of animal facilities, etc.  Room
 
numbers or other unique identifiers should be
 
provided, however it is not necessary to include
 
the location of processing equipment within
 
rooms and areas.  Reference can be made to
 
manufacturing flow information presented in
 
response to section II. C. 2. of this guidance.
  3.  Other Products
 
A comprehensive list of all additional products
 
to be manufactured or manipulated in the areas
 
used for the product should be provided.  The
 
applicant should indicate in which rooms the
 
additional products will be introduced and the
 
manufacturing steps that will take place in the
 
room.  An explanation should be given as to
 
whether these additional products will be
 
introduced on a campaign basis or concurrently
 
during production of the product which is the
 
subject of the application.  Any additional
 
products that may share product contact
 
equipment with the product in question should be
 
indicated (dedicated vs. multi-use equipment
 
should be delineated for each process step, in
 
this section or other appropriate sections of
 
the application).  A brief description should be
 
provided as to the type and developmental status
 
of the additional products. 

4.	  Contamination Precautions
 
For all areas in which operations for the
 
preparation of cell banks and product
 
manufacturing are performed, including areas for
 
the handling of animals used in production, the
 
following information concerning precautions
 
taken to prevent contamination or cross-

contamination should be provided:
 
	  air quality classification of room or area
 
in which operation is performed, as
 
validated and measured during operations;
 
	  a brief, narrative description of the
 
procedures and/or facility design features
 
for the control of contamination, cross
 
contamination and containment (air pressure
 
cascades, segregation of operations and
 
product, etc.) - this is of particular
 
importance for multi-use areas or for work
 
with live organisms;
 
	  general equipment design description, eg.
 
does design represent an open or closed
 
system or provide for a sterile or non-

sterile operation, and; 

	  a description of the in-process controls
  performed to prevent or to identify
 
contamination or cross contamination.
 
The manipulation of more than one cell line in a
 
single area, or the use of any piece of
 
equipment for more than one cell line, should be
 
indicated and measures to ensure prevention of
 
cross contamination should be discussed.
 
 
C. Method(S) of Manufacture
 
1. Raw Materials and Reagents
 
A list of all components used in the manufacture
 
of the drug substance, and their tests and
 
specifications or reference to official
 
compendia should be provided.  For purchased raw
 
materials representative certificates of
 
analysis from the supplier(s) and/or
 
manufacturer's acceptance criteria should be
 
included in the submission.   Process gases
 
(e.g., air, carbon dioxide) and water are
 
considered raw materials. 

A list with tests and specifications of all
 
special reagents and materials used in the
 
manufacture of the drug substance, e.g., culture
 
media, buffers, sera, antibiotics, monoclonal
 
antibodies, preservatives, should be submitted. 

In cases where an ancillary biological product
 
is used in the manufacture of the drug substance
 
(e.g., a monoclonal antibody used in affinity
 
chromotagraphy), a detailed description of the
 
preparation and characterization of the reagent
 
should be submitted (Reference 7, 10). 

A description of the tests and specifications
 
for materials of human or animal source that may
 
potentially be contaminated with adventitious
 
agents, e.g., mycoplasma, Bovine Spongioform
 
Encephalopathy (BSE) agent for bovine derived
 
products, and other adventitious agents of human
 
and animal origin should be submitted. 

Validation data or certification supporting the
 
freedom of reagents from adventitious agents
 
should be included in the submission.
  2.  Flow Charts
 
A complete visual representation of the
 
manufacturing process flow should be provided. 

This flow chart should indicate the step in
 
production, the equipment and materials used,
 
the room or area where the operation is
 
performed (may reference the simple diagram in
 
II. B. 2.) and a complete list of the in-process
 
controls and tests performed on the product at
 
each step.  This diagram should also include
 
information (or be accompanied by a descriptive
 
narrative) on the methods used to transfer the
 
product between steps, i.e. Sterile, steam-in
place (SIP) connection, sanitary connection,
 
open transfers under laminar flow units etc. 

Such transfers should be described for movement
 
of product between equipment, areas/rooms,
 
buildings and sites.  Manufacturing steps which
 
are computer controlled should be identified. 

References can be made to other sections of the
 
application for more detailed process
 
information. 

3.	  Detailed Description
 
a.	  Animal Sources 
 
The information submitted concerning
 
animals used in production, such as mice
 
used for ascites or transgenic animals,
 
should include detailed descriptions of the
 
following:
 
	  source of animals;
 
	  method of creating and the genetic
 
stability of transgenic animals;
 
	  adventitious agent screening and
 
quarantine procedures used to assure
 
animals are appropriate for use in
 
manufacturing;
 
	  animal husbandry procedures, and;
 
  veterinary oversight. 
More detailed guidance in these areas may 
be obtained from the ?Points to Consider in 
the Manufacture and Testing of Monoclonal 
Antibody Products for Human Use and the 
?Points to Consider in the Manufacture and  Testing of Therapeutic Products for Human
 
Use Derived from Transgenic Animals.
 
b.	  Cellular Sources
 
i.	  Cell Substrate / Host Cell /
 
Expression Vector System
 
A.  Recombinant DNA Products
 
including rDNA-Derived Monoclonal
 
Antibodies. 

The submission should include a
 
detailed description of the host
 
cell and expression vector system
 
and their preparation as
 
delineated below: (See References
 
9, 10, 12)
 
I.	  Host Cells
 
A description of the source,
 
relevant phenotype, and
 
genotype should be provided
 
for the host cell used to
 
construct the biological
 
production system. The
 
results of the
 
characterization of the host
 
cell for phenotypic and
 
genotypic markers, including
 
those that will be monitored
 
for cell stability, purity,
 
and selection should be
 
included.
 
II.  Gene Construct
 
A detailed description of
 
the gene which was
 
introduced into the host
 
cells, including both the
 
cell type and origin of the
 
source material, should be
 
provided.  A description of
 
the method(s) used to
 
prepare the gene construct
  and a restriction enzyme
 
digestion map of the
 
construct should be
 
included.  The complete
 
nucleotide sequence of the
 
coding region and regulatory
 
elements of the expression
 
construct, with translated
 
amino acid sequence, should
 
be provided, including
 
annotation designating all
 
important sequence features. 

 
  III.  Vector
 
Detailed information
 
regarding the vector and
 
genetic elements should be
 
provided, including a
 
description of the source
 
and function of the
 
component parts of the
 
vector, e.g. origins of
 
replication, antibiotic
 
resistance genes, promoters,
 
enhancers.  A restriction
 
enzyme digestion map
 
indicating at least those
 
sites used in construction
 
of the vector should be
 
provided. The genetic
 
markers critical for the
 
characterization of the
 
production cells should be
 
indicated.
 
IV.  Final Gene Construct
 
A detailed description
 
should be provided of the
 
cloning process which
 
resulted in the final
 
recombinant gene construct. 

The information should
 
include a step-by-step
 
description of the assembly
  of the gene fragments and
 
vector or other genetic
 
elements to form the final
 
gene construct.  A
 
restriction enzyme digestion
 
map indicating at least
 
those sites used in
 
construction of the final
 
product construct should be
 
provided.
 
V.  Cloning and Establishment of
 
the Recombinant Cell Lines.
 
Depending on the methods to
 
be utilized to transfer a
 
final gene construct or
 
isolated gene fragments into
 
its host, the mechanism of
 
transfer, copy number, and
 
the physical state of the
 
final construct inside the
 
host cell (i.e. integrated
 
or extrachromosomal), should
 
be provided.  In addition,
 
the amplification of the
 
gene construct, if
 
applicable, selection of the
 
recombinant cell clone, and
 
establishment of the seed
 
should be completely
 
described. 
 B.  Monoclonal Antibodies. 
A detailed description of the 
development of the monoclonal 
antibody should be provided 
including characterization of the 
parent cells, donor history for 
human cells, immunogen, 
immortalization procedures, 
screening, and cell cloning 
procedures.  (See Reference 7, 9) 
ii.  Cell Seed Lot System 
A.  Master Cell Bank (MCB) 
  
A detailed description of the 
preparation and testing of the 
MCB, as outlined below and in the 
ICH guideline ?Analysis of the 
Expression Construct in Cells 
used for Production of R-DNA 
Derived Protein Products, should 
be submitted. 
The MCB should be described in 
detail, including methods, 
reagents and media used, date of 
creation, quantity of the cell 
bank, in-process controls, and 
storage conditions.  The results 
of the characterization of the 
MCB for identity and purity using 
appropriate phenotypic  markers 
such as morphology, auxotrophy, 
isoenzyme, etc. should be 
included.  Restriction enzyme 
analysis and DNA sequencing data 
supporting the integrity of the 
introduced genetic sequence and 
data supporting the stability of 
both the host cell and final gene 
construct  during storage should 
also be submitted.  For bacterial 
cells, the results of tests for 
contamination with  both lytic 
and lysogenic bacteriophages and  non-host microorganism(s) should
 
be included.
 
The testing of the MCB for 
endogenous and adventitious 
agents (e.g. Murine retroviruses, 
Epstein-Barr virus, mycoplasma, 
bacteria, fungi, other viruses 
and/or virus-like particle), as 
appropriate, as outlined in 
"Points to Consider in 
Characterization of Cell Lines 
used to Produce Biological 
Products", 1993, and ?Points to 
Consider in the Manufacture and 
Testing of Monoclonal Antibody 
Products for Human Use should be 
described.  If new Master Cell 
Banks are to be generated by 
transfer of final DNA construct 
to host cells or by expansion of 
an existing MCB or Working Cell 
Bank (WCB), then acceptance 
criteria for both the new cell 
bank and the drug substance(s) 
produced by the new bank should 
be described.  In particular, 
documentation of the fidelity of 
the introduced nucleotide 
sequence in the new MCB and 
restriction mapping analysis 
should be submitted. 
B.  Working Cell Bank
 
 
A  detailed description of the
 
preparation and testing of the
 
WCB such as those outlined in the
 
applicable guidance documents
 
(References 7, 9, 10, 12) should
 
be submitted. 

The production of the Working
 
Cell Bank should be described in
 
detail, including methods,
 
reagents and media  used, date of
 
creation, quantity of the cell
  bank, number of cell doublings
 
from the MCB and storage
 
conditions.  If there is no MCB,
 
the results of the
 
characterization of the WCB
 
should be provided in the format
 
detailed for the MCB (Section II.
 
C. 3. b. ii.).
 
C.  End of Production Cells (EPC)
 
 
A detailed description of the 
characterization of the EPC that 
demonstrates that the biological 
production system is consistent 
during growth should be provided. 
The results of the analysis of 
the EPC for phenotypic or 
genotypic markers to confirm 
identity and purity should be 
included.  This section should 
also contain the results of 
testing supporting the freedom of 
the EPC from contamination by 
adventitious agents.  The results 
of restriction enzyme analysis of 
the gene constructs in the EPC 
should be submitted.  Further 
guidance can be obtained from the 
ICH document on ?Analysis of the 
Expression Construct in Cells 
Used for Production of R-DNA 
Derived Protein Products and 
?Points to Consider in the 
Manufacture and Testing of 
Monoclonal Antibody Products for 
Human Use. 
    iii.  Cell Growth and Harvesting
 
A detailed description of the process
 
of inoculation, cell growth and
 
harvesting should be submitted.  The
 
composition of the medium, equipment
 
preparation and sterilization, as well
 
as fermentation medium sterilization,
 
should be described.  For all stages
  of any fermentation process the
 
procedures which prevent contamination
 
with adventitious agents should be
 
described. 

The stages of cell growth should be
 
described in detail including the
 
selection of inoculum, scale-up for
 
propagation, and established and
 
proposed (if different) production
 
batch size.  All operating conditions
 
and in-process controls should also be
 
described and appropriate ranges for
 
operating and control parameters, such
 
as fermentation time, cell doubling
 
time, cell culture purity, cell
 
viability, pH, CO2, etc., 

established.  If induction is required
 
for production of protein, detailed 

information including induction
 
conditions and controls employed
 
should also be described. 

The submission should include the
 
process used to inactivate cells
 
utilized in the production of a drug
 
substance prior to their release into
 
the environment.  For cell lines
 
meeting the criteria of Good Large
 
Scale Practice (GLSP) organisms (July
 
18, 1991 FR notice, Vol. 56, no 138,
 
p. 33174), which do not require
 
inactivation prior to release into the
 
environment, the information
 
supporting their qualification as GLSP
 
organisms should be provided.  A
 
description of the procedures used, in
 
the event of a contamination, to
 
inactivate a GLSP culture prior to
 
release should be included.
 
If the culture supernatant or cell
 
pellet is stored prior to processing,
 
data supporting its stability during
 
storage should be provided.
 
The manipulation of more than one cell
 
line in a single area or the use of
  any piece of equipment for more than
 
one cell line should be indicated and
 
measures to ensure prevention of cross
 
contamination should be discussed.
 
c.  Purification and Downstream Processing
 
A detailed description of the purification
 
and downstream processing, including a
 
rationale for the chosen methods, and the
 
precautions taken to assure containment and
 
prevention of contamination or cross
 
contamination should be provided. 

In-process bioburden and endotoxin limits
 
should be specified where appropriate.  Any
 
reprocessing using a validated reprocessing
 
method and the conditions for batch
 
eligibility should be described. 

If applicable, indication (or reference to
 
II. B. 2. ) should be made as to the multi
use nature of areas and equipment (e.g.
 
campaigning vs. concurrent manufacture;
 
dedicated vs. shared equipment) used for
 
these procedures.  A brief description of
 
the controls employed to ensure segregation
 
and prevent cross contamination, or
 
reference to another section containing
 
this information, should be provided.
 
4.  Batch Records
 
A completed (executed) representative batch
 
record of the process of production of the drug
 
substance should be submitted.
  D.  Process Controls
 
1.	  In-Process Controls
 
A description of the methods used for in-process
 
controls, e.g., those involved in fermentation,
 
harvesting and downstream processing, should be
 
provided.
 
2.	  Process Validation
 
A description and documentation of the
 
validation studies should be provided.  If the
 
process was changed or scaled up for commercial
 
production and involved  changes in the
 
fermentation steps, the re-validation of cell
 
line stability during growth should be
 
described, as in the previous section, and the
 
data and results provided.
 
a.	  Validation Studies for the Cell Growth and
 
Harvesting Process. 

A description and documentation of the
 
validation studies which identify critical
 
parameters to be used as in-process
 
controls, to ensure the success of routine
 
production should be submitted.  Reference
 
may be made to the flow diagram(s) as
 
appropriate.
 
b.	  Validation Studies for the Purification
 
Process. 
   
A description and documentation of the
 
validation of the purification process to
 
demonstrate adequate removal of extraneous
 
substances such as chemicals used for
 
purification, column contaminants,
 
endotoxin, antibiotics, residual host
 
proteins, DNA, and viruses, where
 
appropriate, should be provided.  (See
 
references 4, 7 - 10)
      c.  Microbiology. 

A description and documentation of the 
validation studies for any processes used 
for media sterilization, inactivating cells 
prior to their release to the environment, 
if such inactivation is required, etc., 
should be provided.  If the drug substance 
is intended to be sterile, information 
should be submitted as described in the 
?Guidance for Industry for the Submission 
of Documentation for Sterilization Process 
Validation in Applications for Human and 
Veterinary Drug Products. 
E.  Reference Standard(s)
 
1.  Primary Reference Standard
 
If an international reference standard (WHO,
 
NIBSC) or compendial reference standard (USP)is
 
used, submit the citation for the standard and a
 
certificate of analysis.  If no biological
 
potency or chemical reference standard exists,
 
and the applicants establish their own primary
 
reference standard, a description of the
 
characterization, and specifications of the
 
standard should be provided.  Submit the results
 
of testing, such as physicochemical and biologic
 
activity determinations, of the standard and
 
provide a certificate of analysis.  The Standard
 
Operating Procedures (SOPs) to be used for
 
qualifying a new reference standard should be
 
included.  Information should also be provided
 
on the stability of any reference standard. 

2.  Working Reference Standard (if used).
 
If an in-house working reference standard is
 
used, a description of the preparation,
 
characterization, specifications, testing and
 
results should be provided.  The data from the
 
calibration of the in-house working reference
 
standards against a primary reference standard
 
should also be submitted. 
 F.  Specifications / Analytical Methods
 
1.  Drug Substance Specifications and Tests.
 
a.	  Specifications and analytical methods used
 
for release testing, shelf life and
 
distribution should be described. 
 
Specifications and tests for the drug
 
substance sufficient to assure its
 
identity, purity, strength and/or potency,
 
as well as lot-to-lot consistency should be
 
submitted. (See references 3, 4, 7 - 11,
 
13)  Validation of the analytical systems
 
and the data should be provided for non
compendial methods to demonstrate the
 
system suitability.
 
b.	  Certificates of Analysis and Analytical
 
Results
  
Certificates of analysis and analytical
 
results for at least three consecutive
 
qualification lots of the drug substance
 
should be submitted.
 
2.	  Impurities Profile. 
 
A discussion of the impurities profiles, with
 
supporting analytical data, should be provided. 

Profiles of variants of the protein drug
 
substance (e.g., cleaved, aggregated,
 
deamidated, oxidized forms, etc.), as well as
 
non-product related impurities (e.g., process
 
reagents and cell culture components), should be
 
included.
 
G.	  Container/Closure System(S)
 
A description of the container and closure system,
 
and its compatibility with the drug substance should
 
be submitted.  The submission should include
 
detailed information concerning the supplier,
 
address, and the results of compatibility, toxicity
 
and biological tests. Alternatively, a Drug Master
 
File (DMF) may be referenced for this information. 

If the drug substance is intended to be sterile,
  evidence of container and closure integrity for the 
duration of the proposed expiry period should be 
provided. 
H.  Drug Substance Stability 
A description of the storage conditions, study 
protocols and results supporting the stability of 
the drug substance should be submitted in this 
section.  (Refer to ICH document ?Stability Testing 
of Biotechnological/Biological Products or other 
FDA documents such as " Guideline for Submitting 
Documentation  for the Stability of Human Drug and 
Biologics" for specific information.) 
Data from tests to monitor the biological activity 
and degradation products such as aggregated, 
deamidated, oxidized, and cleaved forms should be 
included, as appropriate.  Data supporting any 
proposed storage of intermediate(s) should also be 
provided. 
III.  DRUG PRODUCT 
A.  Composition, including components. 
A tabulated list of all components with their unit 
dose and batch quantities for the drug product or 
diluent in accordance with the ?Guideline for 
Submitting Documentation for the Manufacture of and 
Controls for Drug Products should be submitted. 
The composition of all ancillary products that might 
be included in the final product should be included. 
B.  Specifications & Methods for Drug Product 
Ingredients 
1.  Drug Substances Including All Active Ingredients 
and Ancillary Components.  
This section should contain a description of 
tests and specifications for all active 
ingredients, if not specified in the Drug 
Substance section.  The specifications for all 
ancillary products that are included in this 
product should be provided.  2.  Excipients:
 
Information on all excipients including process
 
gases and water should be included.
 
a.  Compendial Excipient(s). 

A list of compendial excipients and the
 
citations for each should be submitted.
 
b.  Non-Compendial Excipient(s).  

Tests and specifications should be
 
described.  For a novel excipient, the
 
description should include its preparation,
 
characterization, and controls.  For
 
inactive ingredients of human or animal
 
origin, certification, results of testing
 
or other procedures, or validation data
 
demonstrating their freedom from
 
adventitious agents should be provided.
 
C.  Manufacturer(s)
 
The name(s) and address(s) of all manufacturers
 
involved in the manufacture and testing of the drug
 
product including contractors, and a description of
 
the responsibility(ies) of each should be submitted. 

A list of all other products (research &
 
development, clinical or approved) made in the same
 
rooms should be provided.  See II. B. 3. of this
 
document for detailed guidance.
 
D.  Methods of Manufacture and Packaging
 
A complete description of the manufacturing process
 
flow of the formulated bulk and finished drug
 
product should be provided.  This discussion should
 
include a description of sterilization operations,
 
aseptic processing procedures, lyophilization, and
 
packaging procedures.  Accompanying this narrative,
 
a flow chart should be provided that indicates the
 
production step, the equipment and materials used,
 
the room or area where the operation is performed
 
(may reference the simple diagram in II. B. 2.) and
 
a listing of the in-process controls and tests
 
performed on the product at each step.  This flow
  diagram or narrative should also include information
 
on the methods of transfer of the product between
 
steps, i.e. Sterile, SIP connection, sanitary
 
connection, open transfers under laminar flow units,
 
etc.  Such transfers should be described for
 
movement of product between equipment, areas/rooms,
 
buildings and sites.   References can be made to
 
other sections of the application for more detailed
 
process information. 

E.  Specifications & Test Methods for Drug Product
 
1.  Sampling Procedures 
 
The sampling procedures for monitoring a batch
 
of finished drug product should be included.
 
2.  Specifications & Methods.
   
A description of all test methods selected to
 
assure the identity, purity, strength and/or
 
potency, as well as the lot-to-lot consistency
 
of the finished product and the specifications
 
used for the drug product should be submitted. 

Certificates of analysis and analytical results
 
for at least three consecutive batches should be
 
provided. 

   
The validation data for system stability for all
 
non-compendial tests should be provided.  If
 
compendial methods have to be validated to
 
ensure non-interference of special inactive
 
ingredients, the results of those validation
 
studies should be submitted.
 
F.  Container/Closure System(s)
 
A description of the container and closure system,
 
and its compatibility with the drug product should
 
be submitted.  Detailed information concerning the
 
supplier(s), address(es), and the results of
 
compatibility, toxicity and biological tests should
 
be included.  Alternatively, a DMF can be referenced
 
for this information.  For sterile product, evidence
 
of container and closure integrity should be
 
provided for the duration of the proposed expiry
 
period.
  G.  Microbiology
 
Information should be submitted as described in the 
?Guidance for Industry for the Submission of 
Documentation for Sterilization Process Validation 
in Applications for Human and Veterinary Drug 
Products. 
H.  Drug Product Stability
 
A description of the storage conditions, study
 
protocols and results supporting the stability of
 
the drug product should be provided.  This should
 
include information on the stability of intermediate
 
fluids or formulated bulk under specified holding or
 
shipping conditions, as appropriate.  For products
 
administered through pumps or other such delivery
 
devices, data on the stability of the drug product
 
in the delivery system should be provided. 

Stability data supporting the proposed shelf-life of
 
the reconstituted drug product and for all labeled
 
dilutions should be included.  The results of all
 
tests used to monitor biological activity and the
 
presence of degradation products such as aggregated,
 
deamidated, oxidized, cleaved, etc. forms of the
 
drug substance should also be included.  (See
 
references 3, 7 - 13)
 
IV.  INVESTIGATIONAL PRODUCT/FORMULATION
 
A discussion of any differences in formulation,
 
manufacturing process, or site between the clinical
 
trials materials and commercial production batches of
 
drug substance and drug product should be submitted.  If
 
there are differences, a complete description of these
 
differences should be included.  If an investigational
 
drug formulation was different from that of the to-be
marketed finished product, data to support comparability,
 
bioequivalence and/or pharmacokinetic equivalence of the 

two formulations should be provided, if appropriate.  If
 
the manufacturing process and/or site was different, data
 
from appropriate testing to assess the comparability of
 
the investigational and commercial products should be
 
provided (See reference 6).
 
V.  ENVIRONMENTAL ASSESSMENT
  An environmental assessment should be prepared as
 
outlined in 21 CFR Part 25.  This submission should
 
include a  description of the action that is being
 
considered and should address all the components involved
 
in the manufacture and disposal of the product.  A
 
statement of exemption under a Categorical Exclusion may
 
be provided if applicable.
 
VI. 	 METHOD VALIDATION
 
Provide information as described in the ?Guideline for 
Submitting Samples and Analytical Data for Methods 
Validation. 
VII.	  REFERENCES
 
Guidelines
 
1	  Guidance for Industry for the Submission of
 
Documentation for Sterilization Process Validation
 
in Applications for Human and Veterinary Drug
 
Products 

2	  Guideline on Sterile Drug Products Produced By
 
Aseptic Processing.
 
3	  Guideline for Submitting Documentation  for the
 
stability of Human Drugs and Biologics
 
4	  Guideline for Submitting Documentation for the
 
Manufacture of and Controls for Drug Products
 
5	  Guideline for Submitting Samples and Analytical Data
 
for Methods Validation
 
6	  FDA Guidance Concerning Demonstration of
 
Comparability of Human Biological Products,
 
Including Therapeutic Biotechnology-derived Products
 
Points To Consider
 
7	  Points to Consider in the Manufacture and Testing of
 
Monoclonal Antibody Products for Human Use (1994)
 
8	  Points to Consider in the Manufacture and Testing of
  Therapeutic Products for Human Use Derived from
 
Transgenic Animals, 8/22/95
 
9	  Points to Consider - Characterization of Cell Line
 
used to Produce Biological Products, 7/12/93
 
10	  Points to Consider in the Production and Testing of
 
New Drugs and Biologics Produced by Recombinant DNA
 
Technology, 4/10/85
 
International Conference on Harmonization (ICH) Guidelines
 
11	  Stability Testing of New Drug Substances and Products,
 
10/27/93
 
12	  Analysis of the Expression Construct in Cells used for
 
Production of R-DNA Derived Protein Products, 11/28/95
 
13	  Quality of Biotechnological Products: Stability Testing
 
of Biotechnological/Biological Products, 11/30/95