Workshop Report 23

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Preface

This is the report of the twenty-third of aseries of workshops organised by the Euro-pean Centre for the Validation of AlternativeMethods (ECVAM). ECVAMs main goal, asdefined in 1993 by its Scientific AdvisoryCommittee, is to promote the scientific andregulatory acceptance of alternative methodswhich are of importance to the biosciencesand which reduce, refine or replace the use oflaboratory animals. One of the first prioritiesset by ECVAM was the implementation ofprocedures which would enable it to becomewell-informed about the state-of-the-art of

non-animal test development and validation,and the potential for the possible incorpora-tion of alternative tests into regulatory pro-cedures. It was decided that this would bebest achieved by the organisation of ECVAMworkshops on specific topics, at which smallgroups of invited experts would review thecurrent status of various types of in vitrotests and their potential uses, and make rec-ommendations about the best ways forward(1).

The workshop on Monoclonal AntibodyProduction was held in Angera, Italy, on1922 November 1996, under the chairman-ship of Uwe Marx (University of Leipzig,

Monoclonal Antibody Production

The Report and Recommendations of ECVAM Workshop 231,2

Uwe Marx,3 M. Jim Embleton,4 Ren Fischer,5 Franz P. Gruber,6 UlrikaHansson,7 Joachim Heuer,8Wim A. de Leeuw,9 Ton Logtenberg,10

Wolfram Merz,11 Daniel Portetelle,12 John-Louis Romette13 and DonaldW. Straughan14

3Institute of Clinical Immunology and Transfusion Medicine, Department of MedicalBiotechnology, University of Leipzig, Delitzscher Strasse 141, 04129 Leipzig, Germany;4Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road,Manchester M20 9BX, UK; 5Laboratorium fr Biochemie I, ETH-Zentrum,

Universittsstrasse 16, 8092 Zurich, Switzerland;

6

FFVFF, Biberlinstrasse 5, 8032 Zurich,Switzerland; 7Swedish Fund for Research without Animal Experiments, GamlaHuddingevagen 437, 12542 lvsj, Sweden; 8ZEBET, BgVV, Diedersdorfer Weg 1, 12277Berlin, Germany; 9Department of Animal Experiments, Veterinary Public HealthInspectorate, Ministry of Public Health, Welfare and Sport, 2280 HK Rijswijk, TheNetherlands; 10Department of Immunology, University Hospital Utrecht, Heidelberglaan100, 3584 CX Utrecht, The Netherlands; 11INTEGRA Biosciences GmbH, Ruhberg 4, 35463

Fernwald, Germany; 12Department of Microbiology, Faculty of Agronomy, 6 AvenueMarchal Juin, 5030 Gembloux, Belgium; 13Laboratoire de Gnie Cellulaire, Universit de laMditerrane, CESB/ESIL, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France;14FRAME, Russell & Burch House, 9698 North Sherwood Street, Nottingham NG1 4EE,UK

ATLA 25, 121137, 1997 121

Address for correspondence: Dr U. Marx, Institute of Clinical Immunology and Transfusion Medicine, Depart-ment of Medical Biotechnology, University of Leipzig, Delitzscher Strasse 141, 04129 Leipzig, Germany.

Address for reprints: ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (VA), Italy.1ECVAM The European Centre for the Validation of Alternative Methods. 2This document represents theagreed report of the participants as individual scientists.


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Germany). The aim of the workshop was toevaluate the present status of in vitro meth-ods for monoclonal antibody (mAb) produc-tion, and to compare the advantages anddisadvantages of the in vitro methods with

those of the traditional in vivo (malignantascites) method. The workshop participantsassessed various in vitro culture systems forthe propagation of hybridoma cells in termsof: a) the antibody production capacity; b)the concentration, yield and quality of themAbs produced; and c) the capital and run-ning costs of operation. The participants feltthat there are already several scientificallysatisfactory in vitro methods which are bothreasonably and practicably available. Asthese are of moderate cost, and can be shownto be either better than, or equal to, theascites production method in terms of anti-body quality, they concluded that the in vivo

production of mAbs is no longer necessary,except in rare cases where it is alreadyapproved for clinical applications. In thisrespect a guideline on mAb production wasdiscussed at the workshop, and a proposedguideline is included as an Appendix to thisreport. Differences between national policiesand legal controls in several European coun-tries on ascites production were identified,and recommendations have been made to tryto increase the routine use of in vitro meth-ods by mAb producers and users. The spe-cific conclusions and recommendations madeduring the workshop are summarised in thefinal section of this report.

Introduction

Monoclonal antibodies are antibodies whichhave a single, selected specificity and whichare continuously secreted by immortalisedhybridoma cells. A hybridoma is a biologi-cally constructed hybrid of a mortal, anti-body-producing, lymphoid cell, and amalignant, or immortal, myeloma cell. Fol-lowing the discovery of hybridoma technol-ogy in 1975 (2), developments in mAbproduction and in their application have had

profound implications not only on medicalresearch, diagnosis and therapy, but also onbiology in general. Hybridoma technologyrepresents a significant advance because, inprinciple, it provides a means for obtainingunlimited supplies of highly specific antibod-ies.

In the production of mAbs, animals (gen-erally rats or mice) first have to be immu-nised with the target antigen to obtainmortal antibody-producing cells. The biolog-ical construction of hybrids, and the selec-

tion of hybridomas which produce antibodieswith the desired specificities, are carried outin vitro. In the early days of hybridoma tech-nology (the late 1970s), the hybridomasdeveloped in vitro were injected into the peri-toneal cavity of an animal so that usefulamounts of the desired mAb could be har-vested from the ascitic fluid. This procedurewas considered necessary at the time, sinceno efficient large-scale in vitro methods wereavailable. By the mid-1980s, there werealready serious doubts regarding the neces-sity of such a painful animal procedure. Nev-ertheless, as a result of its early introductionas part of the hybridoma technology, ascites

production of mAbs is now employed world-wide, in spite of the ongoing developmentof in vitro technologies and the growing pub-lic pressure to replace or reduce animalexperiments. The urgent need for experts todisseminate information and make recom-mendations about antibody production, tak-ing animal welfare issues into consideration,was recognised by ECVAM in holding aworkshop on avian antibodies in March 1996(3) and, subsequently, in organising thisworkshop on mAb production.

Hybridoma Technology

There are essentially two stages in the pro-duction of mAbs: a) the induction of antibody-producing lymphoid cells in vivo and theselection of antibody-producing hybridomacells in vitro; and b) the in vitro/in vivo prop-agation of selected hybridoma clones. Thefirst stage, the formation and selection of thehybridoma clone, involves the use of one ormore animals (except in rare cases when ahuman mAb is being developed), and is car-ried out in the following way:

1. The antigen is injected into mice (orrats). The antigen is often injected in

combination with an adjuvant, toenhance the immune response, eventhough the use of adjuvant generallyleads to severe side-effects.

2. After an appropriate interval (521 days),the immunised animals are killed and

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lymphoid cells (including progenitor anti-body-producing cells) are isolated fromthe spleen.

3. The lymphoid cells are fused with myelomacells which have been grown in vitro.

4. The two original cell types and thenewly formed hybrids are cultured in aselective medium, such as HAT (hypox-anthine/ aminopterin/thymine) medium,which only allows the hybridoma cells togrow.

5. The supernatant media from the numer-ous in vitro microcultures exhibiting arecognisable growth of hybridomas arescreened for secretion of the desired anti-body, by using various immunoassay pro-cedures.

6. The selected cells are subcultured invitro, using special cloning procedures toensure that each in vitro culture consistsof hybridomas with a single antibodyspecificity only.

7. Hybridoma cells can be cryopreserved atthis stage.

The second stage, the propagation of clonedhybridoma cells, can be accomplished eitherby continuing to grow the cells in vitro, or bypropagating them in vivo in the form ofascites tumours.

Current Demand for MonoclonalAntibodies

The applications of mAbs are numerous anddiverse. They are extensively used in funda-mental research, medicine and biotechnol-ogy. At present, four user groups can beidentified, according to the amount of anti-body required. These are summarised in Fig-ure 1.

User group A: < 0.1g

Approximately 60% of the mAb users inEurope fall within this group, as do many ofthe current users of the in vivo (ascites)method. Small amounts of antibodies areproduced for use in fundamental and appliedresearch, the commercial production of spe-cial diagnostic kits for research, and for ana-lytical purposes.

ECVAM Workshop 23: monoclonal antibodies 123

Figure 1: Monoclonal antibody user groups

D

< 1%

C

~10%

B

~30%

A

~60%

> 10g

0.510g

0.10.5g

! #$%g

amount of

monoclonal

antibody

required


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User group B: 0.10.5g

This group accounts for approximately 30%of mAb users and encompasses a significantnumber of people still using the in vivomethod. Antibodies in these amounts are

required for the development and productionof a wide range of in vitro diagnostic kits andreagents, as well as for evaluating the use-fulness of novel therapeutic mAbs in animalexperiments.

User group C: 0.510g

In this group, which accounts for approxi-mately 10% of mAb users, adoption of the invivo method is comparatively rare. ThemAbs produced are used in routine diagnos-tic procedures and in preclinical evaluationstudies. They are usually produced by largebiotechnology companies but, during the last

few years, the production of these mAbs hasincreasingly been contracted out to smallerfacilities.

User group D: > 10g

Users in this group, who require mAbs forprophylactic and therapeutic purposes invivo, make up less than 1% of all mAb usersin Europe. The mAb production processesthey use are first developed and validated bythe pharmaceutical industry, and are thensubmitted to a regulatory body for approval.

The extensive use of the ascites method by

groups A and B can be attributed to its sup-posed economic advantage as well as to alack of inclination to adopt the new tech-niques. Most of the mAbs produced bythese groups are not used in clinical studiesand therefore do not have to comply withthe standard requirements for pharmaceu-tical products. This has led to a lack ofawareness in these user groups of the dis-advantages of ascites production, such asthe potential for infection by animalviruses, and the reduced immunoreactivityof the mAb due to contamination with non-specific animal immunoglobulins.

Monoclonal Antibody ProductionInVivo

The in vivo procedures entail the use of miceor rats. Initially, the immune systems of theexperimental animals are suppressed (12

weeks before the intraperitoneal [i.p.] injec-tion of hybridoma cells) by injection (i.p.) ofa primer, such as pristane (2,6,10,14-tetra-methylpentadecane) or Freunds incompleteadjuvant. The hybridoma cells then multiply

in the peritoneal cavity, and the ascitic fluidwhich forms is a very rich source of thesecreted antibody.

When an adequate amount of ascites hasformed, the animal is killed and the asciticfluid is collected. Sometimes, the ascitic fluidis first tapped or drained from the peri-toneal cavity while the animal is underanaesthetic, with a second and final harvestbeing taken once the ascites has reformed.The mAb product can be harvested 521 daysafter the injection of hybridoma cells.

Approximately 5ml of ascites can be obtainedfrom a mouse, and 1040ml from a rat. Thus,for the production of a mAb with a given

specificity, it may be necessary to use one ormore mice, depending on the amount of anti-body required.

The main advantage of the ascites methodis the extremely high yield of antibody,which generally lies in the range 120mg/ml.In addition, the method is not excessivelylabour-intensive.

However, these advantages are out-weighed by a number of disadvantages. Themain disadvantage of the ascites method isthat it is extremely painful for the animalsused, due to the following: a) the injection ofprimer; b) the resulting peritonitis caused bythe primer; c) abdominal tension; and d) the

invasive tumours which result (46). Properanimal husbandry facilities are mandatory.The mAbs produced generally show areduced immunoreactivity of 6070%, asopposed to an immunoreactivity of 9095%for antibodies produced in vitro, due to cont-amination by biochemically identicalimmunoglobulins. There is also a potentialrisk of product contamination by viruseswhich are pathogenic to humans. A furtherdisadvantage is that the individual batchesof harvested ascitic mAb are of variable qual-ity, and they are contaminated with bioreac-tive cytokines.

In Vitro Production Procedures

In vitro production systems

During the last 20 years, a wide range ofin vitro production systems have been

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developed for different purposes. While mostof them are useful for the in vitro productionof mAbs, they differ in terms of: a) the easewith which they are handled; b) the antibody

yield per culture or bioreactor run; and c) the

maximum antibody titre achievable. Theantibodies produced generally express animmunoreactivity of 9095%, irrespective ofthe system used.

Three categories of in vitro production sys-tem can be identified according to the princi-ple underlying the culture system: a) staticand agitated suspension cultures; b) mem-brane-based and matrix-based culture sys-tems; and c) high cell density bioreactors.Some of these systems have been reviewedrecently (7, 8).

Static and agitated suspension culturesSystems in this category, which include the

widely used T-flasks, roller cultures andspinner cultures, allow the growth of a max-imum of two litres of supernatant per cul-ture unit, and a maximum antibody yield of100200mg. They are easy to handle in cellculture laboratories, enable varioushybridoma cell lines to be propagated simul-taneously, and are useful for most of theusers in group A.

Investment costs are low because dispos-able plasticware is readily available, particu-larly when using T-flasks. The use ofserum-free media, or low-cost additives per-mitting a reduction in the serum concentra-tion, can greatly reduce costs, whileefficiently supporting hybridoma growth(913). For example, two serum-free mediause a combination of transferrin and insulin(9, 10), whereas two low-serum media use acombination of 1% fetal calf serum (FCS)and 0.1% Primatone, a peptic digest of ani-mal tissues. This supports hybridomagrowth in all culture methods tested at leastas efficiently as 5% FCS, at approximately25% of the cost (M.J. Embleton, personalobservation).

For the production of mAbs in amountsgreater than 100mg, conventional stirredtank bioreactors of different sizes are avail-

able. These bioreactors need to be used byspecially trained staff and are relevant foruser groups B, C and D.

The concentration of hybridoma cells insuspension cultures hardly ever exceeds 5 106 cells/ml and, in general, the maximumantibody concentration achievable is below

100g/ml. As a result of the low antibodyconcentration, the supernatant usually hasto be concentrated by ultrafiltration if anyfurther purification steps are to be carriedout.

Feeding of cultures may be carried outperiodically if required but, in practice, anti-body concentration is increased by 24 timesif the cultures are allowed to grow to exhaus-tion over 23 weeks without feeding.

Membrane-based and matrix-based culturesystemsThis category includes membrane-based andmatrix-based static cultures as well as sus-pension bioreactors. These systems are suit-able for user groups A, B and C, whichrequire up to 10g of mAb.

In membrane-based systems, the cells arecultured in compartments separated from

the nutrient supply by perfusion mem-branes; special gassing membranes enhancethe oxygen transfer into these systems. Theyproduce yields of up to 100mg per culture(user group A), and generate intermediateantibody concentrations of up to 500g/ml.In addition, they are easy to handle andenable various different cultures to be runsimultaneously in routinely equipped cellculture laboratories.

In matrix-based systems, such as fluidisedbed or ceramic bioreactors, the immobilisa-tion of cells on matrices enables them to beperfused actively and continuously withfresh medium. Irrespective of the size andrunning time of the bioreactors, 0.110g ofmAbs (user groups B and C) can be pro-duced, corresponding to a maximum concen-tration of 500g/ml. In most cases, thesupernatant produced has to be concentratedby precipitation or ultrafiltration before spe-cial purification procedures can be carriedout. Special training is required for theproper handling of these systems.

High cell density bioreactorsThis category includes all culture systemswhich are capable of generating cell densitiesgreater than 108 cells/ml and which, in cer-

tain cases, can maintain viable tissue-likecultures. The bioreactors meet the needs ofuser groups B and C, as they are capable ofgenerating 0.110g mAb. The correspondingconcentrations lie in the range 0.55mg/ml,due to the high cell densities in these sys-tems. They can be run in conventional cell

ECVAM Workshop 23: monoclonal antibodies 125


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culture laboratories and models are availablefor the simultaneous propagation of differentcell lines. The product can be used directly orpurified without prior concentration. Train-ing is recommended for these systems and is

usually provided by the manufacturer.In the most common system within thiscategory, the hollow fibre bioreactor, the cul-ture medium is passed through bundles ofhollow fibres, enabling the cell growth com-partment to be perfused continuously andeffectively. Due to the high antibody concen-tration, the maximum amount of 500mg ofantibody needed by user group B can be pro-duced in a bulk of only 500ml of supernatant,which is easy to handle and process in a con-ventional cell culture laboratory. Even foruser group C, which requires up to 10g ofmAb, the total product can be produced inonly 10 litres.

The different categories of culture systemare listed according to their usefulness to thedifferent user groups in Table I. Instead ofthe maximum achievable mAb concentra-tion, the concentration which is normallyachievable is given. The types of systems rec-ommended for the different user groups, onthe basis of their ease of handling, produc-tion costs, and advantages with respect toantibody purification, are highlighted.

In vitro process development

Several problems are associated with the useof serum-containing media for the in vitroproduction of mAbs, the most importantbeing the high protein content which makesantibody purification either difficult orimpossible. Other problems are animal wel-fare concerns relating to the production offetal serum, its cost, its uncontrollable vari-ability in quality from one batch to another,and the risk of its contamination by viruses,mycoplasma and unsuspected prions (14).

All commercial companies with a longexperience of cell culture, and many smallnew biotechnology groups, now offer variousserum replacements from bovine plasma andserum substitutes, and ready-to-use serum-free media which may contain many serum-

derived proteins (~3mg/l) or reducedamounts of essential proteins (~30g/ml), orwhich may be devoid of proteins and pep-tides. Potentially important supplements arealso supplied separately to fortify and opti-mise basal versions of the classical mediacurrently used (15, 16).

Hybridoma growth and mAb production inserum-free media are variable processeswhich depend on the physical and nutritionalrequirements of: a) the specific hybridomacell line; b) the complexity of the serum-free

formulation; and c) the culture conditions ofthe bioreactors (17). Therefore, during theweaning process by which a subpopulation ofcells is adapted to growth in a new environ-ment, one needs to optimise criteria such asthe cell growth rate, the maximum cell con-centration, the final mAb concentration, andthe quality of the mAb and its productionrate. It is also necessary to ensure that theselected subpopulation exhibits the sameimmunoreactivity as the population whichwas cultured in the presence of serum (16).

In most cases, the use of an optimisedserum-free formulation rather than a serum-containing medium offers two advantages: a)

the mAb is produced in greater yield andwith less expense (16, 18); and b) subsequentdownstream processing is facilitated.

Monoclonal antibody quality

Both monoclonal and polyclonal immuno-globulin G (IgG) antibodies are N-glycosy-lated at amino acid 297, a conservedasparagine (Asn) residue in the second con-stant domain of the heavy chain (CH2).Human serum IgG might be associated withat least 30 different biantennary complexoligosaccharides (19), but these representonly 25% of the antibodys molecularweight.

Under physiological conditions,N-glycosy-lation at Asn 297 plays an important role inseveral biochemical processes: a) the fixationof complement C1q (17, 20; G. Winter, A.R.Duncan & D. Burton, patent numberPCT/GB88/002111); b) the binding of Fc-receptors; and c) the resistance of the anti-body to proteolysis. In addition, biologicallyimportant processes, such as phagocytosis,antigen-dependent cellular cytotoxicity, andthe clearance and placental transfer ofmAbs, can be influenced by the type,sequence and structure of their glycosyla-tion.

In addition to glycosylation at Asn 297,glycosylation also occurs, in very rare cases,in the variable region of mAbs (21). If suchan additional glycosylation is present on amAb, it may influence its antigen bindingcapacity, with the result that the respectivehybridoma clone is unlikely to be picked out

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TableI:Appr

opriateculturesystemsforthefourmonoclonalantibodyuserg

roups

Usergroup

Categoriesof

culturesystem

A

B

C

D

0.1g(~60%ofusers)

0.1

0.5g(~30%ofusers)

0.5

10g(~10%ofusers)

10g(

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