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PRODUCTION AND QUALITY CONTROL OF

MONOCLONAL ANTIBODIES

Gu id e li n e Ti t le P ro du c t ion An d Qu ality Co n tro l Of Mo n o c l o n a l

A n t i b o d i e s

Le g is la t ive bas i s D ire ct ive 75/318/EEC as am e nde d

D a te of fi rs t a d o pt io n S e e p re v i ou s t it le s /o t h e r r e fe r e nc e s

This vers ion adopted De cember 1994

Date of en try i n to

f o r c e

July 1995

Status Las t re v ise d De ce m be r 1994

P r e v i o u s t i t l e s / o t h e rr e f e r e n c e s

Orig inal ly pub l i shed as two gu ide l ines : Produc t ion and Qual i ty Con tro l o f Huma n Monoc lonal An t ibod ies ( J u l y

1990) and Produc t ion and Qual i ty Con tro l o f Monoc l ona l

Antibodies of Murine Origin (June 1987) . The prev ious

referenc e of the combine d vers io n w as III /5271/94

Ad di t io na l N ote s Th is g uide lin e o ut l in es t he re qu ire me nts fo r m u ri ne ,

h u m a n an d e n g i n e e re d m o n o c l o n a l a n t i bo d i e s f o r

t h e ra p e u ti c ( i n c lu d i n g e x v i v o a p p l i c a t io n ) a n d i n v i v o

d iagnos t ic use in humans . I t concerns the app l icat ion o f

Part 2, sect ions A, B, C, D and E of the Annex to D ir e ct iv e

75/318/EEC as ame nded with a vie w to the grant ing of a

market ing author isation for a new me dic inal product .

CONTENTS

1. IN TROD U CTION

2 . P OIN TS TO CON S ID E R IN MAN U F ACTU R E

3. SOURCE CELLS

4 . C EL L L IN E P R OD U CI NG TH E MON OC LON AL AN TIB OD Y

5. CELL LINE PRODUCING THE RECOMBINANT MONOCLONAL ANTIBODY

6. CELL BAN K SYSTEM

7. C HAR AC TE R IS AT ION O F T H E MON OC LON AL AN TI BOD Y

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PRODUCTION AND QUALITY CONTROL OF

MONOCLONAL ANTIBODIES

1. IN TROD UCTION

In this document the requirement s for m ur ine, h uma n and engineered m onoclona l

an tibodies for ther apeutic (includin g ex vivo applicat ion) an d in vivo dia gnostic use i n

hu ma ns ar e out lined. Monoclonal an tibodies inten ded for u se in the pur ificat ion of other

products should be shown to be pure an d free from advent itious agent s by the met hods

described. Monoclona l an tibodies to be used for diagn ostic pur poses in vitro ar e not the

concern of this note for guidance.

Monoclonal antibodies are antibodies with a defined specificity derived from cloned cells ororgan isms . They can be obta ined from im mort alised B lymphocytes that ar e cloned a n d

expanded as cont inuous cell lines (mu rin e an d hu ma n m onoclonal an tibodies) or fr om

rDNA-engineered mammalian or bacterial cell lines (engineered monoclonal antibodies).

Importan t considera tions for the clinical use of monoclona l an tibodies include the possible

un intent iona l imm un ological cross-reactivity of the antibody with hu man tissue an tigen s

oth er t han th ose desired, and t he possible presence of virusesin the products.

1.1 Mo n o c lo n a l an t i bo d ie s o f m u r in e o r i gi n

Murin e monoclona l an tibodies are obtain ed from mur ine hybridoma s produced by fusion of

B-lymphocytes from immunised mice or rats with murine myeloma cells.

A general problem with the ther apeutic use of mu rin e monoclona l an tibodies in ma n is the

possible indu ction of an tibodies in the recipient again st mu rine imm un oglobulin (h u m a n

a n t i m urine a ntibody or HAMA response). This may result in adverse reactions and limit

the dur at ion of effective an tibody ther apy. In a ddition t he in vivo ha lf life of mu r in e

monoclonal ant ibodies is relatively short. It may be prudent to m inim ise the mu rin e protein

load administered to the patient by the use of a parental myeloma cell lines which does not

itself synthesise immunoglobulin chains.

1.2 H u ma n m o n oc lo n al a n ti bo d ie s

The advant ages of hum an m onoclonal ant ibodies over mu rine monoclona l ant ibodies a re

that hu ma n r ecipients ar e less likely to develop an tibodies against them (although an ti-

idiotypic and possibly anti-allotypic ant ibodies ma y st ill be produced) an d that h u m a n

an tibodies ar e likely to ha ve the full r an ge of biological fun ctions, such as those of the F c

region which may be species specific. There may be other advantages such as selection of a

subclass of ant ibody with pa rt icular properties.

Murin e monoclona l ant ibodies are almost always prepared using cell l ines (hybr idoma s)

ma de by fusion of lymphocytes from a n im mu nised donor with myeloma cells. Th is is n ot

the case for hum an mon oclonal an tibodies as, despite encoura ging ear ly r eports, ther e i s

s ti l l no really sat isfactory h uma n myeloma fusion partn er. As a result , a major difficult ywith the production of hu ma n monoclona l an tibodies has been the genera tion of hyb r idoma

lines of acceptable sta bility. It is also difficult in ma ny cases to obtain a nt igen-pr imed

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lymphocytes suita ble for fusion. In view of th is, a nu mber of alterna tive stra tegies h ave been

devised for production of human monoclonal antibodies. These are:

a ) Fus ion of human lymphocytes (usual ly per iphera l b lood or lymph-node der ived) with a

mu rine m yeloma or hybrid hu ma n-mur ine myeloma line. This procedure is

essentially s imilar to the hybridoma technique used to produce mur ine m onoclona l

an tibodies, but present s some techn ical p roblems in that a lower fusion efficiency i s

usually found and human chromosomes are lost preferentially. This procedure may be

regar ded as a compr omise due to the absence of a suita ble hu ma n myeloma fus ion

partner .

b) Transformat ion of human lymphocytes wi th Epste in-Barr v irus (EBV). This procedure

has been used for many years to produce continuous, rapidly growing human B cells.

c ) Fus ion of human B-lymphocytes wi th a human lympho-blas toid B-ce ll line.

d) Fus ion of an EBV-transformed human B-lymphocyte l ine wi th a mouse myeloma ce l l

l i n e .

Other meth ods for gener at ing stable lines secreting hu ma n an tibodies ma y be developed or

exploited in futu re.

1.3. E n g in e e r e d m o n o c l o n a l a n ti b od i e s

An alternative approach to circumvent the HAMA response, the limited duration of effective

mu rine ant ibody therapy and several ma nu factu ring problems in the production of hu m an

monoclona l an tibodies is the production of so called chimer ic an d hu ma nised m onoclon a l

an tibodies usin g recombina nt DNA (rDN A) technology and euk ar yotic gene expression

meth ods. Both types of rDN A-engin eered monoclona l an tibodies contain hu ma n s equen ces.In chimeric ant ibodies th e variable heavy and l ight chain domains of a hu man ant ibody a r e

replaced by th ose of a rodent (usually mur ine) antibody, which possesses the desired a n t igen

specificity. In h um an ised a nt ibodies only the thr ee short hypervar iable sequ ences

(complemen ta rit y det erm ining regions or CDRs) of the rodent var iable domain s for each

chain a re engineered int o the variable d oma in framework of a hum an ant ibody producing

mosaic variable regions. Humanised antibodies contain a minimum of rodent sequence.

Suita ble cells for expression of the rDN A monoclonal an tibody genes are ma m ma lian cell

lines such as im mu noglobulin non-producing m yeloma cell lines, that ar e capa ble of high-

level expression of exogenous heavy and light chain genes and the glycosylation,

assemblage and secretion of functional antibodies.

Engineered monoclonal antibodies may have the advantages of decreased immunogenicity,

enhanced in vivo circulating half life in combination with optimised specificity and effector

functions.

Certain a spects of the cont rol requirem ent s likely to apply to rDN A derived chimeric a n d

hu ma nised monoclonal an tibody usa ge will be simila r to those alr eady described for

products derived by rDNA technology (Note for guidance Production and Quality Control of

Medicinal Products derived by rDNA Technology) with which the applicants should be

fam iliar . These cont rol requirem ent s concern e.g. statu s of the rDNA with in the host cell,

expression regulation and stability of the expression system and the purification procedure.

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2. P O IN TS TO CON S ID E R IN MAN U F ACTU R E

Several of the requ iremen ts r elatin g to esta blishment s in which biological products ar e

manufactured (e.g. WHO technical Report series 822, 1992: Annex 1 Good Manufacturing

Pr actices for Biological P roducts) apply to the ma nu factu re of monoclonal an tibodies.Addit iona l in form at ion can be foun d in WHO tech nical Report Series 822, 1992: Ann ex 3

Guidelines for Assu rin g the Qua lity of Monoclonal Antibodies for u se in H u m a n s.

Manu factu rer s should also refer to the EU Guide for Good Manu factur ing Pr actice for

Medicinal Products. Attention is drawn to the following points.

2.1. P r o du c t io n p r oc e s s

Man y of the genera l requ irem ent s for the qua lity cont rol of biological products, such a s

potency, abnormal toxicity testing, freedom from contaminants, stability and freedom from

detectable levels of an tibiotics will a pply to m onoclona l an tibodies. It is un desir able to us e

agent s which ar e known to provoke sensitivity rea ctions in cert ain individu als such as, forexample, penicillin or other beta-lactam antibiotics.

2 .2 . B i o lo g i c al m a t e r ia l s u s e d i n t h e p r o d u c t io n

Any rea gent s of biological or igin (e.g. sheep eryt hr ocytes, foeta l calf seru m, bovine s er u m

albumin , huma n tr an sferr in, in sulin, trypsin) used in the generation of the m onoclona l

an tibody producing cell line a nd/or dur ing r out ine production, should be free of m icr obia l

contamination such as mycoplasma, fungi and bacteria. Special consideration should be

given to possible viral contamination and tests for relevant viruses should be performed, e.g.

tr ypsin should be tested for porcine par vovirus.

Bovine sera should be checked and found negative for potentially dangerous viruses (at least

bovine diarrhoea virus, infectious bovine rhinotracheitis and parainfluenza 3). In addition,

bovine sera and other bovine derived biologicals should comply with the requirements in the

note for guidance Minim ising the Risk of T ransmitting Agents causing S pon giform

Encephalopathy via Medicinal Products.

The following points, set out below, should de considered.

3. S OURCE CELLS

Whenever possible, murine tissue and animals used as source materials should be shown to

be free of viruses as indicated in Annex I (a), table 2.

Monoclona l an tibodies obtained from hum an cells present particular concerns r ega r din g

safety. Huma n m onoclonal an tibodies for u se in h uma ns are curr ently u nique in that they

are often derived from cells which are likely immortalised by the deliberate introduction of

EBV, a potential human pathogen. They are likely to be obtained from a transformed human

cell line which is potentially oncogenic. Evidence of contamination with viruses originating

from the donor is cause for concern, as they will by definition be viruses capable of infecting

hu ma ns. Cells from hu ma n origin should be shown free of viruses in dicat ed in Annex I (a)

ta ble 3.

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3.1 Ch a r a ct e r is a t i on o f n o n -s p e c i fi c c e ll s

3 .1 .1 Feeder cel ls

Whenever appropriate, the origin of feeder cells used should be defined. Feeder cells should

be derived from SPF (s pecific p athogen free) animals or cell seed stocks shown to be free of

microbial contamination such as mycoplasma, bacteria and fungi and special consideration

should be given to possible exogenous viral contamination.

3.1.2 Fusion pa rtn er(s)

The fusion par tn er used (e.g. myeloma , hu ma n lymphoblastoid B-cell line) should be fu lly

described and documented. The source, name and characteristics of the parental cell line

should be given. It should be shown that the cell line is a pure cultu re a nd is n ot

cont amina ted with cells of other types. If possible the cell line used a s fusion par tn er should

be selected as one which does not synthesise a ny imm un oglobulin chains . Cryopreser ved

sam ples of the cell line used as fusion par tn er sh ould be ret ained in case retr ospective

investigations become necessary.

3.1.3 Host ce l l for t he express ion of the recombina nt m onoclonal an t ibody

A description of th e star ting host str ain or cell line should be provided includ ing the history

of the strain or cell line, its identification characteristics and potential viral contaminants.

Special at tent ion should be given to the possibility of un inten ded cross-cont am ina tion with

other cell lines or viruses not endogenous to a particular cell line.

The cell line used should not synthesise any endogenous immunoglobulin chains before and

after transfection.

Cryopreser ved sam ples of the host cell line should be reta ined in case retr ospective

investigations become necessary.

3 .2 Ge n e r a t io n a n d c h a r ac t e r is a t io n o f th e s p e c i f ic p a r e n t al c e l l

( m u r i n e a n d h u m a n m o n o c l o n a l a n t i b o d i e s )

The sour ce of th e immu ne parent al cells should be docum ented. If an imm un ogen ha s been

deliberately used, inform at ion on i ts source and preparat ion an d on the im m u ni sa ti on

procedure should be provided.

If the immune parental cells are derived from a human donor, information concerning the

health of the donor should be provided. Any relevant clinical data on the donor mu st be

reported, especially data on possible virological infections. Preferably, the description of the

stat e of health of th e donor should cover a period of some month s before and after der ivat ion

to establish that blood born e viruses such as HIV, h epatitis B and hepat itis C were n ot in the

process of incuba tion. If th ese cond itions can not be comp letely fulfilled, th is should be

justified and it should be demonstrated that the cell bank system is devoid of any relevant

viruses (e.g. HIV 1/2, HBV, HCV).

For the production of monoclonal antibodies of major therapeutic value it may be necessary

to use cells potentially contaminated by a virus. In such a case, it will be necessary to look at

the possible detection of the virus in the cell bank and to add one or more steps dedicated to

inactivate this virus in the processing of monoclonal antibody.

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4. CELL LINE PRODUCING THE MONOCLONAL ANTIBODY

4.1. Ge n e r a ti o n o f th e c e l l l in e

A complet e descript ion of the produ ction of the cell line secret ing the monoclon al an t ibody

should be provided includ ing deta ils of cell fusion, EBV tr an sform at ion an d cl on in g

procedures where appropriate. Sufficient data should be given to allow an assessment of the

efficiency of the cloning procedure.

Agents used in the fusion and selection procedure should be described (e.g. PEG).

4 .2 . Ch a ra cter i sa t io n o f th e ce l l lin e

The char acteristics of the monoclona l ant ibody producing cell line should be detailed. These

should include the specificity, class and, where appropriate, subclass of the immunoglobulin

secreted, together with any dis tingu ishing featur es , such as isoen zyme /imm u noche mi calma rker s . The production of imm un oglobulin cha ins originat ing from the fusion part ner

should be determined. The antibody secretion should be stable in respect to both the type of

antibody (class switch) and level of expression up to and beyond the population doublings

used for r out ine production. Appropriate precautions should be ta ken to avoid cross-

contamination with other cells.

5 . CELL LINE P RODUCING THE RECOMBINANT

MONOCLONAL ANTIBODY

5.1 Cl on i n g a n d c h a ra c t e ri s a ti o n o f t h e D N A c o d in g f or t h e n o n -

speci f ic part o f the recombinant mAb

For both chimeric and humanised monoclonal antibody a description of the origin, isolation

and cloning strategy of the heavy and the light chain genes should be provided. In addition

the following information is required:

i ) the in t roduced framework re sidue subs t itu t ions in human ised monoclona l an t ibod ie s

to improve the CDR conformation (where applicable).

ii) a just ification of th e choice of th e imm un oglobulin isotype.

iii) a char acter isat ion of the hu ma n const an t doma in genes (e.g. by res tr ictionendonuclease maps).

5 .2 S e l e c t io n , c l o n in g a n d c h a r a c te r i s at i on o f t h e D N A c o d i n g fo r

the speci f ic part o f the recombinant mAb

The origin of the hybridoma cell line an d the char acter istics of the rodent m on oclona l

antibody used should be described.

A description of the cloning of the rodent heavy and light chain variable region genes from

the hybridoma cell line an d the char acter isat ion of the coding regions of the cloned gen es

should be provided. For humanised monoclonal antibodies a description of the identification,th e meth od of isolation, eith er by cloning or synth esis, an d th e char acterisat ion of the rodent

CDR genes for both heavy an d light chain should be provided.

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5 .3 Co n s t ru c t i on o f th e g e n e c o d i n g fo r th e r e c o m b in a n t m Ab

A descript ion of the str at egy followed eith er to join the rodent var iable fra gmen t to the

human constant region, or, in the case of humanised antibody, to insert the rodent CDR

genes into the human framework region sequences, is required. This documentation should

inc lude:

i ) ce ll l ines a nd vectors used in the genera t ion of the monoclonal ant ibody, and a

description of the expression vectors used for the transfection of rDNA antibody genes

into the ma m ma lian host cell line, in cludin g the origin, str ucture an d s election

m a r k e r s .

i i ) for both the heavy and l ight chain expression vectors the nucleotide sequences of the

genes of inter est an d the flan kin g cont rol regions. A detailed ma p indicat ing the

regions which have been sequenced dur ing const ru ction an d those deduced from the

literat ur e should be given. All the junction r egions creat ed by ligation du r in g

construction should be confirmed by sequencing.

i i i ) a c lear ident i fica t ion of a l l known expressed sequences .

iv ) an ind ica t ion o f any add it iona l modifi ca t ions .

5 .4 Ge n e r a t io n o f t h e c e l l l in e e x p r e s s in g t h e re c o m b in a n t

m o n o c l o n a l a n t i b o d y

In addition to the docum ent at ion concernin g the sta rt ing host str ain or cell line, the

following information is required:

i ) the methods used for in t roducing the vector in to the hos t ce ll , the se lect ion and c lonin g

of the transformants .

i i ) the s t a tus of the vectors with in the hos t .

i i i ) a de ta iled study us ing var ious res t r ict ion enzymes and Southern blot an a lys is

providing convincin g data on the integr ity in the host cell. Useful inform at ion i s

provided on the expression system by Northern blot analysis.

iv ) a detailed description of the s trat egy by which expression of the relevant gene i s

promoted and controlled (during production).

5 .5 Ge n e ti c st ab ili ty

The stability of the host/vector genetic and phenotypic characteristics should be investigated

up to an d beyond the population doubling level or genera tion nu mber expected dur ing fu ll

scale production. Such stability studies should provide detailed information on:

i ) gene copy number in re la t ion to product ivi ty of the cul ture .

i i ) charac ter isa t ion of the monoclonal ant ibody. Analys is a t the protein level and/or DN A

level can be envisaged. Whichever meth od is used, it should be validated an d the

detection limit should be given.

i i i) the level and consistency of expression of both t he heavy and l ight chain.

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6. CELL B AN K S YS TEM

6.1 Esta bl i sh men t o f th e ce l l b a n k sy s tem (MCB a n d MWCB)

It is essent ial that production is based on a well defined cell ban k system. Th is wi ll

norm ally in volve a Master Cell Bank (MCB) and a Manu factu rers Work ing Cell Ba n k

(MWCB). Dur ing the establishm ent of the cell ban k n o other cell lin es should be ha n dled

simu lta neously in t he sam e labora tory suite or by the sam e per sons. The origin, form ,

storage, use, an d details of life expecta ncy at th e ant icipated ra te of use m ust be described i n

full for all cell banks. New working cell banks should be fully characterised.

Samples of the working cell bank should be retained until at least after the expiry date of the

resulting final lot.

6 .2 Co n t r ol o f v i r ol o gi c a l an d m i c r o bi a l c o n t a m i n a t io n

The various cell levels, including MCB, MWCB and PPCB (Post Production Cell Bank; see6.5) should be exam ined for advent itious agent s (vira l, ba cterial, fun gal or m ycopla sm a l).

Special attent ion should be given to viruses which can comm only cont am inat e the species

from which the cell line has been derived. Appropriate tests to demonstr at e the absence of

virus contamination as described in Annex I should be performed.

Certa in cell lines cont ain endogenous viru ses, e.g. ret roviru ses, which may n ot rea dily be

eliminated. Fur therm ore, potential viral conta minat ion m ay ta ke th e form of complete vi r a l

genomes or subgenomic fra gment s resu lting in the expression of infectious vir al par ticles.

Therefore the possibility of mutations of endogenous viruses during prolonged culture should

be considered. The presence of sequences from viral genomes may not disqualify use of the

cells, but an y exogenous vir al nu cleic acid foun d should be iden tified. If theheter ohybridoma ap proach is used for const ru ction of the an tibody secret ing cell line the cell

bank should be examined for the presence of mur ine and hum an viruses.

A cell line which produces any infectious virus capable of infecting human cells would be

acceptable only in exceptiona l circum st an ces. All pr oducts derived from such lines would

ha ve to be cons idered on a case by case basis. If the cell line secretes infectious vir u s,

appropriat e precautions should be ta ken to protect personnel involved in production fr om

infection.

Ther e is special concern about the use of cell lines tr an sform ed by the deliberat e int roduc-

tion of EBV for the produ ction of hu ma n mon oclona l an tibodies. Despite the fact t hat E BV

tr an sform ed h um an B cells in gener al do not secrete viral par ticles th ese cells cont a in

comp lex copies of the vira l genom e an d E BV sequ ences sh ould be sought by PCR or by co-

cultivation with suitable indicator cell lines.

6.3 Ch a ra c te ri sa ti on

A critical part of quality control will involve the full characterisation of cells. The identity

of the cells should be established by distinguishing markers of the cell, such as specific

isoenzyme and immunological features, and phenotypic characterisation.

If the EBV tr an sform at ion procedure is used alone for th e genera tion of a cell line for the

production of hu ma n monoclona l an tibodies difficulties can ar ise in the clonin g procedur e.It is therefore essent ial that ma nu factur ers show convin cing evidence that the cell line i s

monoclonal .

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6 .4 S e cre ti on o f cy to ki ne s

Manufacturers should be aware that lymphocytes and/or feeder cells can secrete a number of

biological mediators which have diverse functions and may cause adverse effects when

adm inister ed to hu ma ns. Considera tion should be given to the ability of the production

process to remove immune mediators such as interferons and other cytokines.

6 .5 E s ta b li s h m e n t o f t h e p o s t p ro d u c t io n c e l l b a n k

For validat ion pur poses a post production cell bank sh ould be esta blished. F or single ha rvest

production 10 or more population doublings beyond the ma ximu m population doubling leve l

used for routine production should be used. For multiple harvest production at a time which

exceeds the total length of the cultivation period by one third is suggested.

7. CHARACTERISATION OF THE MONOCLONAL ANTIBODY

The monoclona l an tibody should be chara cterised th oroughly. This cha ra cter isation m ust

include both biochem ical/physico-chem ical an d b iological/imm un ological properties of the

antibody. In addition the specificity and crossreactivity of the monoclonal antibody should be

assessed.

7.1 B i o c h e m i c a l /p h y s i c o -c h e m i c a l c h a r a c t e ri s a t io n

The biochemical/physico-chemical properties of the antibody should be described in detail. At

least th e following param eters sh ould be determined: class, su bclass (when appropriate) an d

light -cha in composition, molecular weight and either N- an d C-ter min al a min o a cid

sequence, secondar y and tert iar y s tru ctur e.

7 .2 B i o lo g ic a l/i m m u n o lo g ic a l c h a ra c t e ri s a ti o n

The imm un ological properties of the an tibody should be described in deta il. Therefore the

biological/ imm un ological char acterisation should include: a nt igenic specificity i n clu din g

the char act eris at ion of the epitope the an tibody recognises, bin din g capacity, abilit y for

complement binding and activation, cytotoxic properties, antibody dependent cytotoxicity,

ability to modify r elevant a nt igens, capacity to stimu late im mu nocompetent cells a nd the

ability to induce secretion of cytokines or other mediators.

7 .3 S p e c i fi c i ty a n d c r os s r e ac t i vi t y

The ana lysis should include the determina tion of un intent iona l reactivity with or

cytotoxicity for human tissues distinct from the intended target, and cross-reactivity with a

range of human tissues (listed in Annex II) by immunohistochemical procedures.

8. P RODUCTION

In vitro production is the preferred met hod of production. Dur ing the last few years the

technology for in vitro production of monoclonal antibodies has been considerably improved.

In vitro production of monoclona l an tibodies offers a h igh d egree of control a n d

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standardisation and has important advantages over in vivo production with respect to viral

safety, consist ency of production and the absen ce of conta min an t imm un oglobulin s in the

crude harvest. In vitro production in serum free medium is also now feasible. Another

advan ta ge of this method of production is the consider able redu ction of an ima l us age.

Manu factu rer s should be awa re of Directive 86/609/EEC concernin g the protection ofanimals used for experimental and other scientific purposes.

If in vivo production is chosen it must be justified by the manufacturer.

A clear definition of a batch of product for further processing should be provided. A

production batch should nor ma lly origina te from a fresh a mpoule of the MWCB. Det ails of

the cultur e with the in-pr ocess cont rols s hould be pr ovided. Cr iter ia for rejection of the

harvests and premature termination of the culture should be defined.

8.1 In v i tro p ro du c ti on

For each production run, th e presence, extent and natu re of an y m icrobial cont am ina tion i n

the culture vessels immediat ely prior to all h arvesting must be thoroughly exa min ed.

Detailed informa tion to confirm the adequat e sensitivity of the meth ods u sed to detect

cont am ina tion should be provided an d acceptable limits of conta min at ion set. The b u lk

cultu re fluid should be shown to be free from mycoplasm al, mycotic an d bacter ial

cont am ina tion an d should be tested for t he presen ce of viruses using a gener al test

involving inoculation into suitable cell substrates (see Annex I, b).

The comp osition a nd s ource of the cell cult ur e med ium used for pr oduction should be

recorded. If animal serum-derived additives are used, they should be shown to be free from

adventitious agents (See 2.2.).

Ideally not more tha n one cell line should be cultivat ed simu lta neously in the sa m e

production area. If other cell lines are cultivated in parallel, records must be kept of the cell

lines handled and evidence presented for the absence of cross contamination between them.

8.1.1 Single harvest p roduct ion

The maximum permitted generation number for production should be defined and should be

based on inform at ion concern ing the sta bility of the cell line or the up to an d beyond the

level of production. Data on consist ency of growth of the cultu re a nd on the ma int ena nce of

yield with in specified limits should be presented. Appropriate monitorin g of the cell l in e

char acter istics at the end of the production cycles should a lso be un dert aken . E viden ceshould be provided that the yield does not vary beyond defined limits and that the nature and

quality of the product does not change with respect to specific parameters.

8.1.2 Mul t ip le har vest p roduc t ion

The period of continuous cultivation should be specified and this should be based on

information concerning the stability of the system and consistency of the product up to and

beyond this limit. Monitoring of the production system is necessary throughout the life of the

cultu re. The required frequency an d type of monit oring will depend upon several factors

including the nat ur e of th e expression system and monoclona l an tibody, as well a s the total

length of the period of continuous cultivation undertaken. The acceptance of harvests for

further processing should be clearly linked to the schedule of monitoring applied. Evidence

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should be provided th at t he yield does not vary beyond defined limits and th at t he nat ur e a n d

quality of the monoclonal antibody does not change with respect to specific parameters.

8 .2 In v iv o p ro du c ti on

In vivo production should comply to th e additional r equirement s set below.

8.2.1 C hara c te r i sa t ion o f the an im a ls used

The str ain an d origin of the an ima ls used for production sh ould be specified, t ogether with

th eir genotype an d age. They sh ould be from a closed, specific p at hogen-free (SPF ) colon y

which is routinely monitored for those viruses listed in Annex I Table 2. The long term

records of the breedin g colony in respect of freedom from viral cont am ina tion should be

considered in relation to the reliability of maintenance of the colony. Evidence should also

be presented that animals are maintained under SPF conditions at all t imes during transfer

and use.

8.2.2 Harvest and han dl ing of asc it ic fluid

Ea ch production batch should originat e from a fresh a mpoule of the MWCB. Th e m a xi m u m

perm issible nu mber of serial passages in vivo dur ing n orma l production should be defin ed

an d rest ricted: justificat ion of th is limit should include informa tion concernin g the yield of

monoclonal antibody and the stability of the hybridoma characteristics on in vivo passage up

to beyond that used in production. Indefinite passage in animals is not acceptable. A scheme

of priming, inoculation and harvesting should be provided.

The number of animals and the procedure used to prepare the bulk ascitic harvest should begiven in detail. Full details should be provided on any substances used to pre-treat mice or

rat s to facilita te growth of hybridoma s. Description, volume an d concentr at ion of cell

inoculu m should be given. Data concer nin g the titre of the an tibody in an d st ora ge

conditions of the bulk ascitic fluid should be provided (e.g. temperature, duration, details of

an y proteolytic enzyme inhibitors added). Pa rt icular att ention should be paid to the degree

an d na tu re of an y microbial cont am ina tion (bacterial, mycotic and m ycoplas ma l) in the

bulk ascitic fluid. Testing procedur es capa ble of detecting all of the mu rine viruses listed i n

Ann ex I Table 2 should be perform ed, as indicat ed in Annex I(a) and (b), on at least t he first

five bulk harvests of the product. However, it may be expected that general testing methods

for viruses may be sufficient as experience of production is gained. Consequently, after the

first five production runs, general testing for viruses, limited to those described in Annex I

(b), ma y be consider ed adequat e. Any in fectious agent should be ident ified an d tests for

viruses in Group I Table 2 should be negative. If th e sour ce of mice is chan ged to a differen t

colony or supplier, tests described in Annex I(a) should be perform ed on at least t he first five

bulk harvests to re-establish consistency of freedom from contaminant agents.

8 .3 Vi ro l og i c a l a s pe c t s : i n -p r o ce s s c o n t ro l s

The bulk har vest should be tested for the presen ce of viruses using a gener al test in volv in g

inoculation into suitable cell substrates as described in procedures given in Annex I (b).

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9. P U R IF ICATION OF TH E AN TIB OD Y

9.1 Me th od s

Methods used to pur ify the product and their in-process cont rols includ ing their specificat ion

limits should be described in det ail, justified a nd validat ed. It is imp ortan t to ensur e that

pur ificat ion procedures do not impa ir relevan t im mu nobiological feat ur es of the

imm un oglobulin. Pr ocedur es which ma ke use of affinit y chroma tograp hy, for exa mp le

employing monoclonal antibodies, should be accompanied by appropriate measures to ensure

that these substa nces, or any addit iona l potent ial cont am ina nt s a risin g from their use, do

not compr omise the quality a nd safety of the final product. Cross referen ce is ma de to the

note for guidance Virus Validation Studies: The Design, Contribution and Interpretation of

S tudies Validating the In activation and Removal of Viruses.

The criteria for reprocessing of any intermediate or final bulk should be carefully defined,

validated and justified.

Considera tion should be given to incorp orat ing pr ocedur es which in act ivat e/elimi na te

potential viral contaminants where such methods will not compromise the biological activity

of th e product.

9 .2 Va li d at i on o f th e p u r i fi c a ti o n

The capacity of the purification procedure to remove unwanted host cell derived proteins,

nucleic acids, carbohydrates, viruses and other impurities including product-related proteins

should be invest igat ed th oroughly. Any ina ctivat ion process used should be shown to be

effective and not compromise the biological activity of the product. The reproducibility of the

purification process with respect to its ability to remove specific contaminants, should also bedemonstr at ed. Stud ies using, for example, a car efully selected group of viruses which exhibit

a r an ge of physico-chemical featur es relevant t o their beha viour on pu rification (see note for

guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies

Validating the Inactivation and Removal of Viruses ), host-cell proteins, other potential

impu rities der ived from the production process (e.g. heavy or light chain im m u n oglobu lin

fragm ent s) and DNA int ent iona lly m ixed with the cru de prepara tion (spiking) should be

undertaken. The choice of the nucleotide probe to detect DNA contamination should be

relevant to the system used. A reduction factor for such cont am ina nt s at each sta ge of

pur ificat ion, an d overall, sh ould be esta blished by using, if necessary, concent ra tions of

viruses, host cell proteins, other potential impurities and DNA in excess of that expected

during normal production.

Where a cell line cont ain s vir al subgenomic fra gment s (see section 6.2) con sider at ion

should be given to usin g appropriate viral nu cleic acid in DNA s piking studies. Where a

hybridoma line h as been established by tr an sform at ion with Epstein-Barr virus, specific

EBV sequences sh ould be sought by sensitive techniques such as the polymera se ch a in

reac t ion.

Validat ion of the pur ificat ion process should also include just ificat ion of the wor k in g

conditions such as column loading capacity, column regenerat ion and sanitisat ion a n d

length of use of the columns.

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10. Th e b u lk fi n a l p ro c e s s e d pr od u c t

10.1 T h e m o n o c l o n a l a n t i b o d y

Rigorous cha ra cterisat ion of the pur ified monoclonal an tibody by chemical an d b iological

meth ods will be essent ial. At least the following par am eters should be determ ined: cla ss ,

subclass an d light-cha in composition, glycosylat ion patt ern, integr ity of the molecule by

an alysis of the ra tio hea vy/light chain, microheter ogeneity, m olecula r weight , N- an d C-

term inal amino acid sequence, and secondar y and tert iary s tructure of the ant ibody. Wit h

increa sin g experience, the tests for subclass, light chain composition, N- and C- t er m in a l

amino acid sequence and secondary and tertiary structure could be omitted. The total protein

content, the degree of aggregation an d molecular fragment ation of the im mu n oglobu lin

should be deter mined. Appropriate specificat ions for these param eters, with a ccepta nce

limits, should be set. Especially for engineered an d hu ma nised an tibodies su fficien t

sequence inform at ion to char acterise th e gene product adequa tely sh ould be obtain ed by

peptide mapping or amino acid sequencing.

Particular attention should be given to use a wide range of analytical techniques exploiting

differen t physico-chem ical properties of the molecule. Exam ples of suitable techniques ar e:

SDS-polyacrylamide gel electrophoresis under reducing and non reducing conditions,

isoelectr ic focusing, colum n chroma tograp hy (includ ing HP LC), peptide ma pping, a m in o

acid ana lysis , circular dichr oism a nd carbohydrate ma pping. The ma nufacturer should

provide clear photographs of the gels, etc..

The im mu no-rea ctivit y of the an tibody should be ass essed. Th e specific activity of the

pur ified monoclona l an tibody should be det erm ined (un its of activity/weight of product).

A clear differen ce should be ma de between the an alyt ical tests perform ed du r in g

development, in order to fully cha ra cterise the monoclona l an tibody and tests per form ed

rout inely on ea ch batch of pur ified bu lk pr oduct. Qua lity cont rol tests should be car ried out

routinely on each batch of purified bulk product according to the Guide to GMP.

10.2 P u r i ty

Data should be provided on conta min an ts whose presence is ant icipated in the fin a l

processed product. The level of cont am ina tion consider ed as a cceptable should be jus tified,

and criteria of acceptance or rejection of a production batch should be given. It is important

that the techniques used to demonstrate purity be assessed using as wide a range of methods

as possible, includin g ph ysico-chemical a nd imm un ological techniques. These sh ouldinclude tests for vira l a nd cellular nu cleic acid and pr otein conta min at ion of par ent al,

hybridoma, or host cell origin, as well as on materials derived from tissue culture medium

or materials which have been added during the production or purification processes.

Measur emen ts of total protein and cellular DNA concent ra tions, specific a ctivity,

microbiological an d chemical purity should be reported for the fina l product. Assays of

endotoxin level should also be carried out.

10.3 Ad v e n t i t i ou s a g e n t s

The final bulk product should be shown to be free from bacterial, fungal and mycoplasmal

conta min at ion. Evidence should be presented to show that an y vira l cont am ina nt kn own to

be possibly present in the bulk harvest has been eliminated or inactivated (see Annex I).

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11. CONSISTENCY AND ROUTINE B ATCH CONTROL OF BULK

PROCESSED P RODUCT

A compr ehen sive a na lysis of the init ial batches of a product should be un dert ak en to

establish consistency with regard to identity, purity and potency. Thereafter, a more limitedseries of tests may be appropriate as outlined below.

11.1 C on s i s t e n c y

Eviden ce should be pr ovided on the cons ist en cy of pr oduction, for exam ple on a t least fiv e

consecut ive full scale production batches. This should include inform at ion on the bu lk

harvest and final dosage form as well as on in-process controls. In the case of a production

where mu ltiple ha rvests are applied, batches from d ifferent ferm ent at ion r un s ar e n eeded.

The studies should include biological, chemical and immunological methods to characterise

an d assay the monoclona l an tibodies and meth ods to detect an d ident ify im pur ities. An y

differences which occur between batches should be noted.

11.2 Ro u t in e b a tch co n tro l a n a ly s is

11.2 .1 Ident i t y

A selection of th e test s used to char acter ise the pu rified m onoclon al an tibody sh ould be used

to confirm t he product ident ity for each bat ch. The met hods employed should in clude tests for

the biological activity as well as physico-chemical and immunological methods. Engineered

antibody should be subjected to sequence verification of the peptide backbone by adequate

methods such as peptide mapping.

11.2 .2 Puri ty

The degree of purity desirable and attainable will depend on several factors; these include

the nature and intended use of the product, method of its production and purification and also

the degree of cons ist ency of the production pr ocess. The pur ity of each batch should be

established an d be within specified limits. For engineer ed monoclona l an tibodies the

an alysis should include sensit ive and r eliable assays for DNA of host cell origin an d the

vectors app lied to each bat ch of product. Str ict upper limit s should be set for DN A in the

product.

The product should be shown to be free from microbial cont am ina tion. Eviden ce should bepresented to show that an y vira l cont am ina nt kn own to be present in the bulk har vest h as

been eliminated or inactivated (see Annex I). Pyrogenicity should be tested for.

Particular attention should be given to assessment of the degree of aggregation or molecular

fragm ent at ion of the imm un oglobulin. All possible steps should be taken to prevent

aggregat ion. Limits for the presence of oligomeric imm un oglobulin molecules should be

justified.

11.2 .3 Test for po tency

When appropriate, the biological activity of the monoclonal antibody should be established bya biological assay. In addition information on specific activity will be of considerable value

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an d should be reported. A fully char acterised reference pr epara tion is required to

standardise measurements of specific activity (see section 13).

12. SP ECIFICATIONS AND REFERENCE MATERIALS

The studies described in section 10 will contribute to a definitive specification for the product

when justified by th e inform at ion obtained from the exam ina tion of successive batches a n d

results of batch analysis, as indicated in section 11.

The reference prepar at ion sh ould be produced from a su itable batch of the product, which ha s

been clinically evaluated and fully characterised in terms of chemical composition, purity,

potency an d biological activity. Crit eria for esta blishing the reference prepa ra tion a n d

criter ia for re-testing an d pr olongation of th e shelf life should be st at ed.

13. MODIFIED MONOCLONAL ANTIBODIES

The preparation of sub-fragments of the antibody (Fab or F(ab')2 f ragments ) may have

advan ta ges for some applications. Where su ch fragm ent s ar e preferred for clinica l use,

their molecula r an d an tigenic properties should be defined. Appropriate an alyt ical tests

should be perform ed. Specified limits for impu rities such as fragm ent s other th an t hose

desired or inta ct imm un oglobulin , should be defined. Specifications, with limits, should be

given for each contaminant (e.g. residual levels of enzymes used, such as pepsin or papain),

specific activity, immunoreactivity, and antigen cross-reactivity. A reference batch should

be prepared and all assays should be validated.

The thera peutic an d diagnostic uses of monoclona l an tibodies an d a nt ibody subfr agm ent s

can somet imes be enh an ced by chemical modificat ions (e.g. ra diolabelling, conjuga t ionwith a toxin, att achment to specific su bstan ces for ta rget ing or chem ically link ing of two

an tibody molecules or their derivatives to genera te a bispecific an tibody). F or these a

detailed description of th eir prepara tion and pur ificat ion should be supplied. Ea ch re levan t

step in the production process requires validat ion an d qua lity cont rol coverin g sour ce

ma ter ials, limits for impur ities arisin g from the production process, evidence for

consist ency etc. Modifications can chan ge the propert ies of the monoclona l an tibody a n d

general requiremen ts for such products mu st include inform at ion concern ing the biological

ha lf-life of the an tibody, of the m edicin al product or toxin, an d also of the conjuga te aft er

injection int o a recipient. In for ma tion about the specificity, t he toxicity an d st ability of the

conjugate should also be supplied.

Criteria an d specificat ion limits for p ur ity an d potency of the final pr oduct should be applied

and immunoreactivity and antigen cross-reactivity should be determined. Additional

specific control procedures may be required, but these are dealt with best on a case by case

bas is .

The preparation of a reference batch is required and all assays should be validated.

Detailed information for the production of radiolabelled monoclonal antibodies can be found

in the note for guidance Radiopharmaceuticals based on Monoclonal Antibodies.

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14. FINISHED PRODUCT AND DEVELOPMENT

PHARMACEUTICS

The development of the formulation should be described in detail and justified, particularly

with regar d to the presence and am ount of sta bilisers su ch as album in an d/ or deter gents.The pr oduct in final cont ain ers should be shown to comply with the requ iremen ts of the

Eu ropean directives a nd pha rm acopoeias. In circum sta nces where this is not possible the

omission of tests should be justified by the manufacturer.

15. P RODUCT EQUIVALENCE

Some chan ges or adapt at ions in t he production of a monoclona l an tibody dur ing cli n ica l

development or subsequent to product approval can lead to an alter ed form of the an tibody

with identical specificity. Examples of such changes are: transition of in vivo production to

in vitr o production, cha nges in cultu re procedur es or cultu re conditions, chan ges i n

purification procedure, or additional modifications of the monoclonal antibody molecule. In

these cases, studies to prove product equivalence should be performed to show that both forms

of the antibody are essentially identical.

In all cases th ese studies should include a complete physico-chem ical an d biological

characterisation of both antibodies.

15.1 In v i tro s tu d ies o n p ro d u ct eq u iv a len ce

The physico-chemical char acter istics of the monoclona l an tibody, like isotype, su bclass,

microheterogeneity, molecular weight, primary structure, secondary structure, glycosylation

patt ern, stru ctur al integr ity, should be deter mined. The biological chara cterisat ion should

include immu noreactivity and crossreactivity, the determina tion of relevant fun ctiona l

characteristics and binding studies to determine affinity.

When there ar e cha nges in the cell culture procedur e/conditions without cha nges in the

MCB, relevant par am eters su ch as morph ology, cell growth, viability, isoenzymes, a n d

stability of production should be analysed.

15.2 In v iv o s tu d ies o n p ro d u ct eq u iv a len ce

The decision on the selection of in vivo tests depends on the results of the analyticalcha ra cter isation. In t he case of ident ical a na lytical r esults of both form s of an tibody, at least

the pharmacokinetic, biodistribution and half life should be determined.

15.3 Clin ica l s tu d ie s

When both monoclona l an tibodies ar e demonstra ted to ha ve ident ical ph ysico-chem ical,

biological an d pha rm acological char acter istics, clinical stu dies perform ed with the form er

monoclona l an tibody can be accepted. However, an essentia l prer equisite is that the

production is based on the sam e MCB. Otherwise, clinical tr ials ha ve to be carr ied out with

the second form of antibody.

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15.4 Ma n u fa ctu r in g p ro ced u re

Consist ency of the ma nu factur ing procedure of the monoclona l an tibody, in clu di n g

validat ion of the production process and qua lity control in accorda nce to the requ irem en ts

should be demonstrated.

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A NNEX I

Testing for viru ses sh ould be perform ed in laboratories with experience in rout ine vir u s

testing and should be performed in accordance with good laboratory practice.

Table 1 lists the tests for viruses to be performed at the different stages of production.

Table 2 lists viruses which should be consider ed as potential conta min an ts in the

manufacture of monoclonal antibodies produced by cell lines of murine origin.

Table 3 lists viruses which should be consider ed as potential conta min an ts in the

manufacture of monoclonal antibodies produced by cell lines of human origin.

Test ing for viral Contaminat ion

a ) T es t s for de tect ion of speci fi c v iruses

( i ) Monoclonal ant ibodies produced by ce ll lines of murine or igin

Tests for detection of specific viruses listed in table 2, for example Mouse

Antibody Production (MAP) or Rat Antibody Production (RAP) tests or other tests

of at least equivalent sens itivity an d r eliability. Additiona l specific tests m a y

need to be car ried out for lymph ocytic chor iomenin gitis virus (LCMV), mou se

cytomegalo virus, mouse rotavirus (EDIM), thymic virus and lactic

dehydrogenase virus. Tests capable of detecting murine retrovirus should be

included , for examp le the XC plaque a ssay or the S+ L- focus a ssa y for the

detection of ecotropic or xenotropic retrovirus respectively.

(i i ) Monoclonal ant ibodies produced by cell l ines of hum an origin

For hu ma n m onoclonal an tibodies the viruses which ma y be foun d in the cell

line depend to some extent on the nature and health of the donor. They may be

specifically able to infect B lymphocytes. As a m inim um , the viruses which a r e

kn own to persist in lymphocytes an d a re listed in table 3 should be tested for.

Viru ses should be sought by cultu re meth ods employing cell lines in clu di n g

virus free lymphoblastoid cells as well by examination of the lymphocyte line

itself by use of imm un oche mical procedures, electr on m icroscopy, South ern

blotting, polymerase chain reaction or other sensitive techniques.

( i i i ) Engineered monoclonal an t ibodies produced by mamma lian ce ll l ines

For engineer ed m onoclona l an tibodies the viruses which may be foun d in the

cell line depend on the origin of the cell line. Relevant viruses should be tested

for.

b) Inocula t ion of ce ll cul tures capable of de tect ing a wide range of mur ine , hu man, an d,

if relevant, bovine viruses. Examples of useful cell types (substrates) are: murine

fibroblast cultures, e.g. mouse embryo cultures; human fibroblast cultures, e.g. human

diploid cells such as MRC5; continuous cell lines of human, murine and bovine

origin. The indicator cell lines should additionally be tested for haemadsorbing

viruses (with erythrocytes from human blood group O, guinea pig, chicken) at the end

of th e observat ion time. Tests for ret roviru ses should be included.

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c) Tes ts in anima ls for advent it ious agents should include the inocula t ion by the

intr am uscular r oute of each of the following groups of an ima ls with the test m a t er ia l

or with disrupted cells from the seed lot propagated beyond the ma ximu m level (or

population doubling, as appropriate) used for production:

2 li t te rs of suckl ing mice , comprising a t leas t 10 animals less than 24 hours old

10 adu lt mice

5 gu inea -pigs

Test material should be injected intracerebrally into each of 10 adult mice.

The anim als should be observed for at least 4 weeks. Any an imals th at a re sick or sh ow an y

abn orma lity sh ould be investigated to establish t he cau se of illness. Test ma ter ial can be

consider ed to be suitable for production if at least 80 % of the anim als inoculat ed r em a in

healt hy and su rvive the observat ion per iod and none of the an ima ls shows evidence of the

presence in the tested material of any adventitious agent.

Fertilised eggs may also act as useful substrates. Test material should be injected into eggs

by appropriate routes, the chorioallantoic membrane, amniotic cavity and yolk sack of each

of 10 embryonat ed chicken eggs, 9-11 days old. The embr yona ted eggs should be exa m in ed

after not less than 5 days incubation. The allantoic fluids should be tested with guinea-pig

and chick or other avian red cells for the presence of haemaglutinins.

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TABLE 1

TESTING SCHEME FOR VIRAL CONTAMINANTS

Annex I s ec t ions which are app l icab le

MCB or MWCB (a ) (b) (c)

Mou se br eedin g Colon y (a )

Ascit ic flu id h a r vest (a )* (b)

In vit ro bu lk ha rvest (b)

Bulk final processed product Specified tests of (b) if virus cont am inat ion was detected in

the bulk ha rvest

* I t i s proposed tha t these tes ts should be carr ied out on a t leas t the fi rs t five product ion

r u n s .

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TABLE 3

HUMAN VIRUSES

V i r u s

Human Immunodeficiency Virus (Type 1, Type 2)

Human T cell Leukaemia Virus (Type I, Type II)

Cytomegalo virus

HHV6

Epstein - Barr virus

Hepatitis B virus

Hepatitis C virus

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ANNEX II

Suggested list of hu ma n tissues to be used for imm un ohistochem ical or cytoche m ical

investigations of cross reactivity of monoclonal antibodies. This list should reflect thespecificity of the antibody and its particular use.

Tonsil, thymus, lymph node

Bone marrow, blood cells

Lung, liver, kidney, bladder, spleen, stomach, intestine

Pan creas, par atid, th yroid, para-thyroid, adrena l, pituita ry

Brain, peripheral nerve

Heart , s triated muscle

Ovary, testis

S k i n

Blood vessels

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ANNEX III

Glossary

1. Mu r i n e

Murine mean s derived from an an imal belonging to the Muridae family which in clude s

mice and rats .

2 . Ce ll Ban ks

a ) Ma st er cell ba nk (MCB)

A homogeneous suspension of the original cells on which production is based, aliquoted into

individua l conta iner s for stora ge (e.g. in a liquid n itr ogen r efrigerat or). The origina l cell

line may not necessarily have been produced by the manufacturer.

For engineer ed pr oducts the cells in the ma ster cell bank ar e alr eady tr an sform ed by the

expression vector conta inin g the desired gene. In some cases it may be necessary to esta-

blish separate master cell banks for the expression vector and the host cells.

b ) M a n u fa ct u r e r s w or k in g ce ll b a n k (M WC B)

A homogeneous suspension of cells derived from the master cell bank(s) by a finite passage

level, aliquoted into individual containers for storage (e.g. in a liquid nitrogen

refr igera tor) .

In both cell banks, all containers are treated identically during storage, and once removed

from storage, the containers are not returned to the cell bank stock.

c ) P os t p rod uct ion cells (P P C)

Post production cells are the cells cultured up to 10 or more population doublings beyond the

maximum population doubling level used for routine production (single harvest production)

or cells cultured for a period of time which exceeds the total length of the cultivation period

by one third (multiple harvest production).

3 . P ro d u c t io n Me th o d

a ) P r od u ct ion a t f in it e pa s sa ge (s in gle h a r ve st )

This cultivation method is defined by a limited number of passages or population doublings

which mu st n ot be exceeded durin g production.

b ) Cont inuous cu l tu re p roduct ion (mul t ip le ha rves t )

The number of populat ion doublings are specified based on in form at ion concern ing the

stability of the system a nd t he consistency of the product criteria for t he t ermina tion has to be

defined by the m anu factur er.

4 . Bu lk Ha rve s t

This is a homogeneous pool of individu al ha rvests, lysates or a scitic fluids which i s

processed in a s ingle manufacturing run.

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5 . B ulk Fin al Ac tiv e S ubsta nc e

This is the fina l product, after completion of the pr oduction process, obta ined from a b u lk

harvest. It is maintained in one or more container s and used in the preparation of the final

dosage form. The generation of this final batch has to be clearly defined and unambiguously

recorded by the producer.

6 . F in ish e d P ro du c t

The active substance is formulated and filled into final, sealed containers which hold the

product in its final dosage form , i.e. th e finished product. The container s of a filling lot a r e

processed together and uniform in their contents and biological potency.

7. En gi ne e re d Mo no clo na l An ti bo dy

A human monoclonal antibody in which critical amino acid residues are replaced by

molecular technology.

8 . Fus ion Partne r

A cell line fused with the antibody producing cell with the intention to immortalise this cell.

9 . Fe e de r Ce lls

Cells or cell line co-cult ivated wit h the an tibody pr oducing cell lin e to pr ovide opt ima l

growth conditions.