Producing an Anti-ß2 Integrin MAb By a Hybridoma Cell Linevivo animal studies before seeking...
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T T he most fascinating aspect of our immune system is its capacity to recognize and dispose of virtually any foreign microorganisms or substances considered as non-self intruders. This modular defense system is largely contributed by the presence in our sera of antibodies (Abs). They potentially interact with an infinite number of molecular structures, so an equivalent number of different specificities is necessary. Abs are immunoglobulins made by the B-cell pool of the lymphatic system. The Ab response is polyclonal, but each B-cell clone makes a homogeneous monospecific Ab population. The use of Abs in medicine goes back to 1890, when Von Behring and Kitasto successfully used Abs from horses to neutralize diphtheria toxin. Only after the mid-1970s, when Kohler and Milstein developed hybridoma technology for making monoclonal Abs (MAbs) in vitro, did they see the potential for successful widespread use (1). MAbs have revolutionized many aspects of biology and medicine. They were first very successful as diagnostic reagents, but their success as therapeutic agents was less pronounced then expected early on. With the growth and development of life-science knowledge and technology, including the emergence of novel concepts underlying the molecular bases of several major diseases — innovations in MAb production (more stable hybridoma cell lines, contaminant-free culture products, and so on) — immunotherapy using Abs is undergoing a renaissance. Indeed, 62 out of the 370 identified biotechnology products under development are therapeutic MAbs; 13 are in phase III studies, and three are undergoing regulatory review (2). A recent survey indicated that 6% of all drugs in development are MAbs (2). Additional MAb candidates are expected to enter shortly into clinical trials. The rapid growth of MAb-based drugs that will need to be manufactured has raised the issue of a possible shortage of manufacturing capacity in the foreseeable future. As a consequence, the pressure is on to improve production technologies and ensure more manufacturing capacity, without the need for new, large, costly manufacturing plants. PRODUCT FOCUS: MONOCLONAL ANTIBODIES PROCESS FOCUS: PRODUCTION WHO SHOULD READ: PROCESS DEVELOPERS, PROJECT MANAGERS KEYWORDS: ANTIBODIES, PROCESS OPTIMIZATION, HYBRIDOMAS, SCALE-UP , PERFUSION CULTURE LEVEL: INTERMEDIATE B IO P ROCESS TECHNICAL 54 BioProcess International DECEMBER 2003 Editor’s Note: When we say biotechnology is a global industry, we’re taking into account many permutations that may or may not be familiar to our readers in the United States, Europe, Japan, and Canada. GMPs may or may not apply, but the fact is that biotech industry is occurring outside those areas covered by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). It is our intention with our “Biotech Around the World” series of articles to increase awareness of such work. n Producing an Anti-ß2 Integrin MAb By a Hybridoma Cell Line in a 2-Liter Stirred-Tank Bioreactor Héla Kallel and Dahmani M. Fathallah
Transcript of Producing an Anti-ß2 Integrin MAb By a Hybridoma Cell Linevivo animal studies before seeking...
TT he most fascinating aspectof our immune system isits capacity to recognizeand dispose of virtually anyforeign microorganisms or
substances considered as non-selfintruders. This modular defensesystem is largely contributed by thepresence in our sera of antibodies(Abs). They potentially interact withan infinite number of molecularstructures, so an equivalent numberof different specificities is necessary.Abs are immunoglobulins made bythe B-cell pool of the lymphatic
system. The Ab response ispolyclonal, but each B-cell clonemakes a homogeneous monospecificAb population.
The use of Abs in medicine goesback to 1890, when Von Behringand Kitasto successfully used Absfrom horses to neutralize diphtheriatoxin. Only after the mid-1970s,when Kohler and Milsteindeveloped hybridoma technologyfor making monoclonal Abs (MAbs)in vitro, did they see the potentialfor successful widespread use (1).MAbs have revolutionized manyaspects of biology and medicine.They were first very successful asdiagnostic reagents, but theirsuccess as therapeutic agents wasless pronounced then expected earlyon. With the growth anddevelopment of life-scienceknowledge and technology,including the emergence of novelconcepts underlying the molecularbases of several major diseases —innovations in MAb production(more stable hybridoma cell lines,contaminant-free culture products,and so on) — immunotherapy using
Abs is undergoing a renaissance.Indeed, 62 out of the 370 identifiedbiotechnology products underdevelopment are therapeutic MAbs;13 are in phase III studies, andthree are undergoing regulatoryreview (2). A recent survey indicatedthat 6% of all drugs in developmentare MAbs (2). Additional MAbcandidates are expected to entershortly into clinical trials. The rapidgrowth of MAb-based drugs thatwill need to be manufactured hasraised the issue of a possibleshortage of manufacturing capacityin the foreseeable future. As aconsequence, the pressure is on toimprove production technologiesand ensure more manufacturingcapacity, without the need for new,large, costly manufacturing plants.
PRODUCT FOCUS: MONOCLONAL
ANTIBODIES
PROCESS FOCUS: PRODUCTION
WHO SHOULD READ: PROCESS
DEVELOPERS, PROJECT MANAGERS
KEYWORDS: ANTIBODIES, PROCESS
OPTIMIZATION, HYBRIDOMAS,SCALE-UP, PERFUSION CULTURE
LEVEL: INTERMEDIATE
B I O P R O C E S S TECHNICAL
54 BioProcess International DECEMBER 2003
Editor’s Note: When we say biotechnology is a global industry, we’re taking intoaccount many permutations that may or may not be familiar to our readers in theUnited States, Europe, Japan, and Canada. GMPs may or may not apply, but the
fact is that biotech industry is occurring outside those areas covered by theInternational Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH). It is our intention with our“Biotech Around the World” series of articles to increase awareness of such work.
n
Producing an Anti-ß2 IntegrinMAb By a Hybridoma Cell Line in a 2-Liter Stirred-Tank BioreactorHéla Kallel and Dahmani M. Fathallah
Hybridoma cell line culture is agood method of generating largequantities of MAbs for laboratoryand diagnostic use. It is a goodmethod for producing the necessaryamount for assessing therapeuticpotential through in vitro and invivo animal studies before seekingapproval from regulatory authoritiesto enter clinical trials and industrialproduction under GMPs.Technologies for growinghybridomas to produce MAbs rangefrom ordinary flasks to spinnerflasks, roller bottles, and bioreactors.Process optimization is notstraightforward, and a specificprocess must be developed for eachMAb-producing hybridoma cell line.Important variables need to beidentified and optimized to ensureprocess consistency and productivity.
In our laboratory, we areinterested in the conceptual use ofMAbs to control physiologicalprocesses such as inflammation andthe immune response itself. Someanti-�2 integrin monoclonalantibodies hold great potential forpreventing inflammation-mediatedtissue injuries in several pathologicalconditions. The Raptiva MAb is ananti-CD11b �2 integrinmonoclonal-antibody-based drugthat has gone through clinical trialsfor the treatment of psoriasis and iscurrently undergoing regulatoryreview. In this work, we focus on theproduction by a hybridoma cell lineof “monoclonal 107,” an antibodythat antagonizes theproinflammatory functions of thehuman leukocyte adhesion moleculeCR3 or CD11b/CD18. MAb 107recognizes, specifically and with highaffinity, the A domain of the CD11b�2 integrin subunit. The aim of ourstudy was to look for the optimalconditions for producing this MAbby growing the hybridoma cell linein a stirred-tank bioreactor
MATERIALS AND METHODS
The murine hybridoma 107 cell lineused throughout this study camefrom M.A. Arnaout at theMassachusetts General Hospital inBoston, MA (www.mgh.harvard.edu). This cell line produces an
IgG2a monoclonal antibody. Cellswere grown in spinner flasks or two-liter bioreactors using RPMI-1640,catalog number 51800-035 fromInvitrogen (www.invitrogen.com)supplemented by fetal calf serum(FCS), catalog number SH30071.03from HyClone (www.hyclone.com)or bovine calf serum enriched withiron (NS), catalog numberSH30072.03 from HyClone.
Spinner flask cultures were carriedout in 250-mL spinner flaskscontaining 200 mL of cultured cells(inoculated with 2 � 105 cells/mL).The cells were grown at 37 °C in a5% CO2 incubator (model 3164,from Forma Scientific). Theexperiments were carried out induplicate. Samples were taken dailyfor cell counts and for themeasurement of glucose, lactate,ammonia, pH, and antibodyexpression levels.
Bioreactor cultures wereperformed in a 2-L bioreactor fromInceltech (a division of NewBrunswick Scientific Co., Inc.,www.nbsc.com) in Toulouse,France. It was equipped with apitched-blade impeller with a spinfilter (5-µm pore size) fixed on its
FFiigguurree 11:: Effect of inoculum viability onhybridoma 107 cell growth in a 2-literbioreactor
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axis. During the cell cultureproliferation step, these conditionsapplied: pH 7.2, pO2 maintained at30–50%, temperature 37°C, andagitation rate 20–30 rpm. Perfusionwas started after two days of culture,and the perfusion rate wasmodulated according to cell density.Samples were taken daily todetermine cell density and viabilityalong with glucose, lactate,ammonia, and antibody levels.
Analytical Methods: The viable andtotal cell concentrations weredetermined using a hemacytometerand the trypan blue exclusion
method. Glucose and lactic acidconcentrations were monitored byenzymatic assays using glucose(catalog number 101-0014) andlactate (catalog number 101-0040)assay kits from Chronolab AG inSwitzerland (www.chronolab.com).Ammonia levels were quantifiedenzymatically by UV-test, catalognumber 171-UV from Sigma(www.sial.com).
Antibody levels were estimatedby an ELISA test using Immulon-2ELISA plates. The plates werecoated with recombinant CD11b A-domain (2 mg/well) at 37 °C forone hour. They were washed oncewith phosphate buffer solution(PBS), then blocked with 1% bovineserum albumin (BSA) at roomtemperature for one hour. Afterwashing three times with 0.05%Tween 20, the plates wereincubated with MAb 107(hybridoma culture supernatantdiluted with PBS and 1% BSA) atroom temperature for one hour.After incubation, the plates werewashed three times and thenincubated with a second MAb (goatanti-mouse IgG conjugated withperoxydase, catalog number M0284from Sigma), at room temperaturefor one hour. Plates were thenwashed three times again andincubated with a peroxydasesubstrate (O-phenylenediamine/H2O2, catalog number P5412 fromSigma) for 30 minutes at roomtemperature. Results werequantitated using an ELISA platereader at 492 nm.
RESULTS AND DISCUSSION
Effect of Serum Type and
Concentration: To optimizehybridoma 107 cell growth, weinvestigated the effect of serum typeas well as serum concentration. Twotypes of serum were tested: fetal calfserum and bovine calf serumcontaining high level of iron andtransferrin (three to four timeshigher than fetal bovine serum). Asshown in Table 1, RPMI 1640supplemented with 10% NS resultedin the highest cell density andantibody production level. Similardata were reported by Kallel et al.(3) for another hybridoma cell line,which produces an anti �2 integrinmonoclonal antibody.
Optimization of Cell Growth andMAb Production in a 2-LiterBioreactor — Effect of InoculumViability: We carried out fourperfused cultures; the bioreactor wasinoculated with 3 � 105 cells/mL.The viabilities of the differentinocula were equal to 66%, 71%,74%, and 75% for cultures 1, 2, 3,and 4 respectively.
During culture 1, we observed asharp decrease of cell density leveland viability one day after theinoculation of the bioreactor (Figure1). The start of perfusion at day tworesulted in a slight increase of celldensity level. However, a dramaticdecrease in cell viability and densityoccurred the following days.
For culture 2, the viability of theinoculum was equal to 71%, celldensity level, and viability remainedconstant for one to two days, andthen their levels fell. For cultures 3and 4, we noticed a cell growth afterthe inoculation of the bioreactor.Two days after the start of theculture, cell density level was equal to0.7 � 106 cells/mL and 1.05 � 106
cells/mL, respectively for culture 3and 4. The start of perfusion at daytwo, enhanced cell growth. At theend of culture, we reached 1.3 �106 cells/mL (culture 3) and 1.6 �106 cells/mL (culture 4).
These data, suggest that theinoculum viability is a criticalparameter for the growth ofhybridoma 107. To growhybridoma 107 cells in a bioreactor,the viability of the inoculum shouldbe higher than 75%.
TTaabbllee 11:: Hybridoma 107 cell growth and monoclonal antibody in different media. Cells weregrown in spinner flasks.
5% NS 10% NS 5% FCS 10% FCS
Cell density/mL 0.73 � 106 1.2 � 106 0.6 � 106 106
Maximum specific growth rate (h-1) 0.026 0.038 0.027 0.031
Mab level (µg/mL) 29 � 2 32 � 1 28 � 2 31 � 3
FFiigguurree 22:: Hybridoma 107 cell growth in a2-liter stirred tank bioreactor underdifferent aeration conditions
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EFFECT OF AERATION MODE
We performed two perfused culturesof hybridoma 107 cells (Figure 2).During the first culture, pO2 was setat 50% and stirring speed at 30 rpm.Dissolved oxygen level wascontrolled by the process controlunit that injects air in the liquidphase when required. CO2 neededfor pH regulation was also injectedin the liquid phase. The secondculture was carried out in the sameconditions except that stirring speedwas set at 20 rpm.
Although both cultures werestarted with an inoculum viabilityhigher than 75%, we observed a celldensity decrease two days after thestart of the culture. Monitoring ofglucose level indicates that thisnutrient is not limiting. Therefore,the decrease of cell density levelobserved is probably due to shearstress and particularly to cell burdendue to continuous direct gassparging.
PERFUSION CULTURE OF
HYBRIDOMA 107 CELLS
To minimize shear stress effect onhybridoma 107 cell growth,agitation rate was reduced to 20 rpm and pO2 at 30%. Moreover,the culture was aerated continuouslyby air injection in the headspace ofthe bioreactor, but direct spargingwas intermittent.
Figure 3 shows that in theseculture conditions, compared withthe previous cultures, cell growthwas enhanced immediately after thebioreactor inoculation. During thebatch culture phase, we reached ahigh cell density level of 1.05 �106 cells/mL. Cell viability wasequal to 90%; residual glucose,lactate, and ammonia levels wereequal respectively to 1 mM, 18.41mM and 1.49 mM. The start ofperfusion at day two resulted in anincrease of cell density level. At dayfour, cell density level was equal to1.75 � 106 cells/mL; residualglucose, lactate, and ammoniaconcentrations had slightlyincreased.
Figure 3 also indicates that fromday six to 12, perfusing the cultureat a constant rate equal to one
reactor volume/day didn’t enhancecell growth or antibodyconcentration. Nevertheless,increasing the perfusion rate to 1.5 reactor volume/day at day 12resulted in an increase of cell densityand monoclonal antibodyproduction levels. At day 15, wereached a cell density level of 1.8 � 106 cells/mL. Antibodyconcentration was equal to30 µg/mL.
The analysis of Figure 3 alsoshows that antibody production andcell density correlate; therefore, itseems that for hybridoma 107 cellline, antibody production isassociated with cell growth.
Environmental and operatingculture factors affect cell growth aswell as antibody production. Amongthese factors, culture aeration isconsidered one of the mostimportant factors. Indeed it’s crucialto provide sufficient oxygen to meetthe cell culture demand. However,hybridoma cell lines like othermammalian cell lines are sensitive to
shear stress induced by agitation andby direct gas sparging. It’simportant to optimize the aerationoperating conditions for optimal cellgrowth. Different authorsdemonstrated the usefulness ofdetermining optimal aerationconditions (agitation speed, aerationmode, gas flow rate) for cell linesstudied (4–6).
As far as the state of theinoculum is concerned, only limitedexamples related to this parameterwere reported in the literature.Varley and Birch (7) reported thatthe state of the inoculum influencescell ability to withstand shear forces.According to the data we obtained,it appears that the state of theinoculum is a critical environmentalfactor that should be consideredduring process optimization.
OPTIMAL CONDITIONS
We showed that in spinner flasks,hybridoma 107 cell growth wasoptimal in the RPMI 1640 � 10%NS medium. We demonstrated that
FFiigguurree 33:: Growth and monoclonal antibody production by hybridoma 107 cells in aperfused 2-liter bioreactor
when using a two-liter bioreactor,inoculum viability and shear forcesaffected cell growth. At the optimalconditions determined, we obtaineda cell density level of 1.8 �106 cells/mL. Monoclonal antibodyconcentration was 32 µg/mL, and specific productivity was 25 µg/106 cells/day.
REFERENCES1 Kohler, G; Milstein, C. Continuous of
Fused Cells Secreting Antibody of PredefinedSpecificity. Nature. 1975, 256(5517):495–497.
2 Lubiniecki, AS. Monoclonal AntibodyProducts: Achievement and Prospects.BioProcessing Journal. 2003, 2(2): 21–25.
3 Kallel, H; et al. Optimization of aHybridoma Cell Line Growth and AntibodyProduction in a Spinner Flask Using aTaguchi Experimental Design. Cytotechnol.2002, 39(1): 9–14
4 Carswell, KS; Papoutsakis, ET.Culture of Human T Cells in StirredBioreactors for Cellular ImmunotherapyApplications: Shear, Proliferation and the IL-2 Receptor. Biotechnol. Bioeng. 2000, 68(3):328–338.
5 Emery, AN; Jan, DC; Al-Rubeai, M.Oxygenation of Intensive Cell-CultureSystem. Appl. Microbiol. Biotechnol. 1995,43(6): 1028–1033.
6 Fizil, A; et al. Large Scale Cultivationof an Anti-D (IgM) Antibody ProducingHuman/Mouse Heterohybridoma. ActaMicrobiol. Immunol. Hung. 1996, 43(4):345–357.
7 Varley, J; Birch, J. Reactor Design forLarge Scale Suspension Animal Cell Culture.Cytotechnol. 1999, 29(3): 177–205. ��
Corresponding author Héla Kallel is aSenior Process Scientist at InstitutPasteur de Tunis, and Dahmani M.Fathallah is a Senior Scientist at InstitutPasteur de Tunis, 13, place Pasteur, PO74, 1002 Tunis, Tunisia, phone: 00 21671 848 903, fax: 00 216 71 791 833,[email protected].
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