A systematic approach to the validation of process control...

A Systematic Approach to the Validationof Process Control Parameters forMonoclonal Antibody Production inFed-Batch Culture of a Murine Myeloma

Enda B. Moran,1* Steve T. McGowan,1 John M. McGuire,2

Janet E. Frankland,2 Israel A. Oyebade,1 Wendy Waller,1 Linda C. Archer,2

Lilla O. Morris,2 Jyoti Pandya,2 Samantha R. Nathan,2 Lee Smith,2

Mervyn L. Cadette,2 Jurek T. Michalowski2

1Biopharmaceutical Process Sciences, GlaxoWellcome Research &Development, South Eden Park Road, Beckehnam, Kent, BR3 3BS, UK;telephone: +44(0)20 8639 6551; fax: +44(0)20 8639 6149;e-mail: [email protected] Analytical Sciences, GlaxoWellcome Research &Development, South Eden Park Road, Beckehnam, Kent, BR3 3BS, UK

Received 6 September 1999; accepted 2 February 2000

Abstract: A systematic approach to the validation of con-trol ranges of control parameters for a cell culture pro-cess producing a monoclonal antibody is described. Spe-cifically, the structure and functional activity of a mono-clonal IgG1 antibody produced at the outer limits ofnumerical ranges of fed-batch culture control parameterssuch as pH and temperature were examined, with theaim of providing assurance that antibody produced un-der varying culture conditions was of consistent qualitybased on a carefully defined set of specifications. An ex-perimental design was created using a half-fractional fac-torial design for fed-batch culture incorporating half ofthe thirty two possible combinations of five selected con-trol parameters at high and low levels. Statistical analy-sis of all data gathered from the study allowed an assess-ment of the effects of the process control parameters ateither high or low outer limits on fed-batch culture re-sponse variables such as growth rate and specific anti-body productivity. Measured values for the responses ofgrowth rate and specific antibody productivity through-out this study ranged from 0.22–0.44 d−1 and 6.4–32 µgmonoclonal antibody/106 cells/d respectively. Analyticalcharacterisation of monoclonal antibody purified fromeach fed-batch culture considered the purity, structureand biological activity of the glycoprotein. All antibodypreparations were identical to each other and to the cur-rent antibody reference standard or control. Glycosyla-tion analysis of certain samples from the study demon-strated that the distribution of glycoforms of the anti-body was not affected by the varying process controlconditions of the fed-batch cultures. © 2000 John Wiley &Sons, Inc. Biotechnol Bioeng 69: 242–255, 2000.Keywords: antibody; fed-batch culture; process controlparameters; process validation; statistical experimentaldesign; glycosylation

INTRODUCTION

Pharmaceutical manufacturers are obliged to provide assur-ance to regulatory authorities that drug products intendedfor clinical trials and commercial distribution are of appro-priate quality, safety and efficacy (Center for Drug Evalu-ation and Research (CDER), 1987; Sykes, 1994). To thisend, process validation exercises are carried out to gatherand document data to provide assurance that a manufactur-ing process consistently and robustly produces a product topre-determined specifications and quality attributes. Thesevalidation tasks routinely involve the qualification of allfactors that may impact on process performance and productcharacteristics including for example, process control pa-rameters, raw materials, personnel, facilities and equipment.

Process validation of a fermentation or cell culture op-eration for the production of a biological therapeutic, in-volves thorough examination of a number of different ele-ments associated with the successful and reproduciblemanufacture of the drug. Equipment and operating stepsmust be demonstrated to consistently produce a crude drugsubstance suitable for progression to downstream process-ing operations (Naglak et al., 1994). The producing organ-ism must be shown to be capable of producing the biologi-cal to a specified quantity and level of quality (structure,biological activity) for the full duration of its intended termin culture (Center for Biologics Evaluation and Research,1997). Identification of critical process parameters influenc-ing process performance, and the development of methodsto control these parameters, are important early activities offermentation and cell culture process development. Valida-tion of the process parameters extends the above studies,and aims to show comparability and ‘fitness-for-use’ ofproduct when produced within certain numerical ranges of* Correspondence to:E. B. Moran

© 2000 John Wiley & Sons, Inc.

the critical control parameters. The range within each pro-cess parameter may routinely vary during production with-out compromising product quality is referred to as the con-trol parameter range (Chapman, 1993). However, validationshould aim to identify the proven acceptable range—a se-lected range of values encompassing the control parameterrange, within which a control parameter may vary withoutaffecting product quality. Hence, if a brief excursion of aparameter outside the control parameter range occurs duringa production run due to equipment failure/operator erroretc., the product may be deemed fit for use if this excursionis within the proven acceptable range. To illustrate theseparameter ranges, consider a cell line producing a recom-binant protein at an optimum pH of 7.5. The proven accept-able range from a validation study might be pH 7.1–7.9. Acontrol parameter range might then be selected between pH7.3–7.7, providing 0.2 pH units of process “safety” outsidethis range.

A whole battery of physico-chemical and functional as-says are now routinely used for the characterisation of bio-logical products. The concept of well-characterised biologi-cals has allowed the biopharmaceutical industry to imple-ment manufacturing or facilities changes during the clinicaldevelopment of a drug product, without the need for addi-tional clinical studies to demonstrate equivalency or com-parability of product pre- and post-change. A testing proto-col specific for monoclonal antibodies might include aminoacid sequencing, tryptic peptide mapping, SDS-polyacryl-amide gel electrophoresis (SDS-PAGE) under reducing andnon-reducing conditions, isoelectric focusing (IEF), size ex-clusion chromatography (SEC), circular dichroism, reversephase high performance liquid chromatography (RP-HPLC), N-terminal amino acid sequencing and bioactivityassays based on complement-mediated cell lysis (CMCL) orantibody-dependent cell-mediated cytotoxicity (ADCC)(Center for Biologics Evaluation and Research, 1997;Schaffner et al., 1995).

Glycoform analysis is also an essential element of mono-clonal antibody characterisation tests, and indeed for allglycoproteins intended for therapeutic use, due to theknown dependability of such glycoprotein characteristics asconformation, bioactivity and pharmacokinetic profile onoligosaccharide structure (Cumming, 1991). Sensitive ana-lytical techniques such as mass spectrometry, RP-HPLC,high pH anion exchange chromatography and capillary elec-trophoresis are now well established for the characterisationof oligosaccharides released from glycoproteins by chemi-cal or enzymatic means (Rudd and Dwek, 1997). HumanIgG1 is N-glycosylated at asparagaine-297 (Asn-297) in theCH2 domains of both heavy chains in the Fc portion of themolecule. Additional oligosaccharide structures or glycansmay be present in the hypervariable region of the Fab do-mains but this is dependent on the presence of N-linkedglycosylation sites. The glycan located at Asn-297 is of thecomplex biantennary type, Figure 1. It is common for aglycoprotein to exist as a heterogenous mix of glycoformsdue to variability in oligosaccharide processing as a protein

is shunted through the glycosylation machinery of the cell.In the case of IgG1, glycoform variability may result fromthe presence or absence of fucose, sialic acids, galactoseresidues or the bisecting N-acetylglucosamine residue onthe Asn-297 N-linked glycan. Structure variation of IgG1glycan may affect both its conformation and bioactivity(Boyd et al., 1995; Wright and Morrison, 1997).

To date, no published information is available describingthe experimental details of process validation of industriallyrelevant cell culture processes. In this report, we describe anapproach to the validation of proven acceptable ranges forcritical process parameters such as pH and temperature forthe production of a humanised monoclonal IgG1 antibodyby a murine myeloma cell line in protein-free fed-batch cellculture. Here, ‘critical’ refers to those process parametersthat must be controlled to ensure the production of IgG1 ofsatisfactory titre, purity and quality. Statistical design ofexperiment (DOE) was used to create a half-fractional fac-torial design for fed-batch culture incorporating multiplecultures controlled at random combinations of the selectedcontrol parameters at either high or low levels in a scaled-down model of a large-scale production process. Statisticaldata analysis allowed the identification of process controlparameters having the most significant impact on the cellgrowth rate, specific antibody productivity and antibodytitres at harvest in this culture system. Comparability ana-lytical testing was carried out on purified antibody to con-firm the consistency of product produced under the varyingculture conditions.

MATERIALS AND METHODS

Cell Line

A recombinant NS0 myeloma cell line transfected with avector encoding the glutamine synthetase (GS) gene (LonzaBiologics, UK) and the heavy and light chain sequences ofa humanised IgG1 monoclonal antibody (hereafter referredto as Mab) was used in this study. Mab was intended for latephase clinical development of a prostate cancer indication.

Figure 1. General structure of human IgG1 glycan linked to asparagine-297 (Asn-297) in the CH2 domain of each heavy chain. Sites of structuralheterogeneity are shown in bold, underlined text. GlcNAc—N-acetylglu-cosamine, Man—mannose, Gal—galactose, Fuc—fucose, SA—sialic acid.

MORAN ET AL.: VALIDATION OF PROCESS CONTROL PARAMETERS FOR FED-BATCH CULTURE 243

Cell Culture Media

Cells were cultivated in a proprietary protein-free growthmedium generally applicable, with minor alterations, to theculture of myeloma, hybridoma, lymphoblastoid and othercell lines for recombinant protein production. The feed me-dium used for the fed-batch studies was a ten-times con-centrated solution comprising a number of components ofthe growth medium previously identified as critical formaintenance of cell viability and antibody production dur-ing fed-batch culture.

Experimental Design

An experimental design was created on DESIGN EXPERT5.0 software (STAT-EASE Inc., US), and used a half-fractional factorial design for fed-batch culture incorporat-ing half of the thirty two possible combinations of fiveselected control parameters at high and low levels, Table I.This type of fractional factorial design allows the assess-ment of the effect of any single control parameter indepen-dent of all others on a particular culture response (specificgrowth rate, specific antibody productivity etc.), eventhough all factors are altered simultaneously during experi-mentation. The high and low levels of control parameters,referred to as the parameter’s outer limits, were as follows:pH 7.1 and pH 7.5, dissolved oxygen tension (DOT) withrespect to medium saturation with air (5% and 30%), seed-ing density (0.27 × 106 cells/mL and 0.43 × 106 cells/mL),temperature (34°C and 38°C) and a pair of culture feedingregimes involving early or late feed medium addition (feedregime A/44 h, 92 h, 140 h; feed regime B/52 h, 100 h, 148

h; all times from culture inoculation). Fed-batch culturesusing feed regimes A or B were harvested at 164 h or 172h respectively i.e. final feed + 24 h. The study was carriedout in four blocks (labeled 1, 2, 3, 4) of four cultures each(labeled A, B, C, D) and used a single revived vial ofGS-NS0 cells from the working cell bank.

Cell Culture

Myeloma cells were routinely maintained by serial passagein 250 mL Erlenmeyer shake flask cultures at 100 mL work-ing volume and incubated at 36°C and 100 rpm in an Innovaorbital shaker incubator (New Brunswick Scientific, UK).Cell inoculum for each block of fed-batch cultures was pre-pared from a stock batch culture maintained for the fullduration of the study by repeated passage to a target celldensity of 0.3 × 106 cells/mL every 72 h in a 7 Lfermenter(FT Applikon Ltd., UK). This inoculum-supply culture wasagitated at 75 rpm by a marine impeller, controlled at pH 7.3by CO2 sparge or 1 M sodium carbonate addition, main-tained at a temperature of 36°C by water-circulation throughthe glass vessel jacket and at a dissolved oxygen tension of$15% with respect to medium saturation with air by directsparging with air. Our small scale cell culture model (3 Lbioreactor) provides a satisfactory indication of cell cultureperformance and productivity at larger scales up to manu-facturing scale at 8000 L, Figure 2. Purified antibody fromthese scales of cell culture (3 L, 80 L & 7000 L) showedcomparative characteristics as determined by SDS-PAGE,IEF, SEC, CMCL assay and glycosylation analysis by RP-HPLC (analytical methods described below), thereby fur-ther verifying this model. Fed-batch cultures were initiatedby inoculating FT Applikon 3 L bioreactors in growth me-dium at 1.5 L working volume at the experimental design-specified seeding densities. The process parameters under

Table I. Half-fractional factorial design for the validation of processcontrol parameters of myeloma fed-batch culture.

Culturereference pH Temperature DOT

Feedregime

Seedingdensity

1A − + + + +1B + − − − +1C − + + − −1D + − − + −2A − − + − +2B + + − + +2C − − + + −2D + + − − −3A − + − − +3B + − + − −3C + − + + +3D − + − + −4A + + + + −4B − − − + +4C − − − − −4D + + + − +

The cultures were controlled at combinations of either high (+) or low(−) limits of each control parameter. In the case of feed regime, + and −represent feed regimes A and B respectively. The numerical values of thehigh and low outer limits of the control parameters are outlined in theMaterials & Methods section.

Figure 2. Growth of Mab-producing myeloma cells in fed-batch cultureusing similar growth and feed media and identical feeding strategies atdifferent bioreactor scales.

244 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 3, AUGUST 5, 2000

investigation were subsequently set to control at either thehigh or low outer limits according to the experimental de-sign. All fed-batch cultures were agitated at 100 rpm bysingle marine impellers. Cultures were fed according to feedregimes A or B with feed medium at 10% v/v (volume offeed/culture volume). Gas supply for the purpose of dis-solved oxygen control was switched from air to a 40:60O2/N2 gas balance at 48 h to cope with the intensive oxygendemand of many of the cultures of the study. Cultures weresampled every 24 h and 30 min post-feed medium additionfor cell enumeration and viability determination and theother analyses described later. Sampling volumes were suchthat the total volume within the bioreactors during fed-batchculture remained at approximately 1.5–1.6 L.

Antibody Purification

The full volume of culture (approximately 1.5 L) was clari-fied by centrifugation at 4000 rpm for 45 min at 4°C fol-lowed by filtration of the antibody-containing supernatantthrough a Sartobran 300 0.45mm/0.22mm (Sartorius AG,Germany). The crude Mab filtrate was first prepared forchromatography by concentrating between 4 to 6-fold usingan Asahi AM-300M hollow fibre unit with a 10 kD mo-lecular weight cut-off rating (Asahi, Japan). Mab was thensequentially delivered to Protein A Sepharose Fast Flowaffinity and SP Sepharose Fast Flow ion exchange columns(both Amersham Pharmacia Biotech, UK) and eluted using0.2 M citric acid, pH 3.5 (Protein A Sepharose FF) and 50mM citrate, 0.2 M NaCl, pH 6.0 (SP Sepharose FF) buffers.The eluted fraction with maximum absorbance at 280 nmwas used for subsequent biochemical analysis. Purified an-tibody sample concentrations typically ranged from 1.7–14mg/mL.

Analytical Methods

Cell numbers and viability were determined by the cell ex-clusion method using erythrosin B staining and countingusing an Improved Neubauer haemacytometer. Averagespecific antibody productivity (qp) was determined from theslope of the best fit regression lines of plots of accumulatedantibody concentration versus the cumulative integral ofviable cells (IVC) throughout fed-batch culture (Bibila etal., 1994; Renard, 1988; Zhou et al., 1997). In a similarmanner, average specific growth rates (mave) were deter-mined from slopes of the best fit regression lines of plots ofcell concentration versus the IVC. In instances where a lagphase occurred in some of the cultures of the experiment,the lag growth data were ignored for the calculation ofmave.The total number of cell generations post revival of theWCB vial for a number of scaling passages in shake flaskculture followed by maintenance of the inoculum by re-peated sub-culturing in the 7 L stock fermenter was com-puted according to:

Np = (t=1

t=p 1

ln 2ln

Xt

Xt8

where Np 4 number of generations (or population dou-blings) afterp passages,Xt 4 number of viable cells at endof passaget, Xt8 4 number of viable cells at start of passaget, t 4 passage number.

Nephelometry

Mab concentrations were determined by nephelometry. AnArray 360 nephelometer (Beckman (now Beckman Coulter),UK) measured the intensity of light scattered by the in-soluble immune-precipitin formed by mixing Mab with ananti-human IgG (anti-Mab) antibody (Beckman, UK) in areaction cell. The change in the intensity of the scatteredlight was proportional to the concentration of Mab in thesample. The quantity of Mab was calculated from a standardcurve constructed using known concentrations of purifiedMab and the measured rates of scatter of light signal at eachconcentration.

Isoelectric Focusing

Isoelectric focusing was carried out on a pH 3.5–9.5 gradi-ent using thin layer Ampholine PAG plate gels on a Mul-tiphor II apparatus (Pharmacia, Sweden). 10mg of Mab wasloaded in each well and focused at 1000 V, 40 mA and 25W for 135 min. The following proteins were used as stan-dards: trypsinogen (pI 9.3), lentil lectin with isoelectricpoints of 8.65 (basic), 8.45 (middle) and 8.15 (acidic), horsemyoglobin with isoelectric points of 7.35 (basic) and 6.85(acidic), human carbonic anhydrase (pI 6.55), bovine car-bonic anhydrase (pI 5.85),b-lactoglobulin (pI 5.20), soy-bean trypsin inhibitor (pI 4.55) and amyloglucosidase (pI3.5). Gels were fixed for 1 h in an11.6% trichloroaceticacid/3.4% sulphosalicylic acid solution, then stained in a hotsolution (60°C) of Coomassie Blue R-250 (0.11%), aceticacid (8%) and ethanol (25%) for 10 min and finally de-stained overnight in a solution of acetic acid (8%) and etha-nol (25%). Band patterns and pI ranges of samples werecompared to the reference Mab standard. Gels were photo-graphed for permanent recording of results.

SDS-PAGE

SDS-PAGE was performed using Pharmacia Excel 8–18%gels on a Pharmacia Multiphor II flatbed system (Pharma-cia, Sweden). Briefly, 5mg of Mab (reduced using dithio-threitol or non-reduced) were loaded on the gel, in additionto two lanes containing BSA at a total amount of 2 ng and4 ng per lane. The gel was electrophoresed at 600 V, 50 mAand 30 W for 75 minutes, stained using a PlusOne™ silverstaining kit (Amersham Pharmacia Biotech, UK) and devel-oped until the 2 ng BSA marker was just visible. Proteinmolecular weight markers were: myosin (212 kD),a2-


macroglobulin (170 kD),b-galactosidase (116 kD), trans-ferrin (76 kD), glutamate dehydrogenase (53 kD), phos-phorylase B (94 kD), bovine serum albumin (67 kD), oval-bumin (43 kD), carbonic anhydrase (30 kD), soybeantrypsin inhibitor (20.1 kD) anda-lactalbumin (14.4 kD) (allAmersham Pharmacia Biotech, UK). Band patterns ofsamples were compared to the reference Mab standard. Gelswere photographed for permanent recording of results.

Size Exclusion Chromatography

Size exclusion chromatography was performed using an iso-cratic Kontron HPLC (BioTek Kontron Instruments, Italy)system with on-line integration. Duplicate injections wereloaded onto a TosoHaas TSK G3000SWXL column(Anachem, UK) which was pre-equilibrated with 0.1 M so-dium sulphate/0.1 M disodium hydrogen orthophosphatebuffer at pH 6.8. Elution was carried out using this buffer ata flowrate of 0.75 mL/min. UV detection at 280 nm wasemployed for the identification of purified peaks. The areasof the peaks attributable to the antibody monomer, aggre-gate and fragments were separately expressed as percent-ages of the sum of the total area under the chromatograph.The elution positions of these impurities were previouslyidentified by spiking Mab reference standard preparationswith known amounts of aggregated and degraded antibody.Degraded antibody (degradation confirmed by SDS-PAGE)was prepared by heating or UV-treating pure Mab prepara-tions. It was assumed that early eluting species of pure Mabpreparations must be attributable to aggregated antibody(aggregation of the preparations was confirmed by SDS-PAGE).

Antibody Binding Activity

Binding activity of Mab was measured using a sandwichELISA. Serial two-fold dilutions of the Mab reference stan-dard preparation and test samples were prepared in PBS andadded to microtitre plate wells coated with Mab’s cell sur-face target antigen, EGP-40. Addition of Protein A-horseradish peroxidase conjugate (Protein A-HRP; Amer-sham Life Sciences, UK) followed by the HRP substrate3,38, 5,58 tetramethyl benzidine (TMB; Sigma, UK) wasused for the detection of bound Mab. The product of theenzymatic action of HRP on TMB was detected at a wave-length of 405 nm. The binding activity of test samples wasobtained by comparing results to the Mab reference stan-dard which had an assigned binding activity expressed inunits per millilitre (U/mL). The specific binding activitywas determined by the following:

Specific binding activity(U/mg) =Binding activity(U/mL)

Mab concentration(mg/mL)

where Mab concentration was determined by nephelometryor the measurement of absorbance at 280 nm.

Antibody Immunoreactivity–Complement-MediatedCell Lysis

LS174T human colon adenocarcinoma cells expressing theEGP-40 antigen (ECACC, UK) were labeled with bis-(acetoxymethyl) 2,28:68,28-terpyridine-6, 69-dicarboxylate(BATDA; Wallac, Finland). BATDA readily diffuses acrosscell membranes and is hydrolysed by intracellular esterasesto the membrane-impermeable moiety, 2,28:68,28-terpy-ridine-6, 69-dicarboxylate (TDA). Mab was added to thecells in the presence of normal human serum as a source ofcomplement. Complement is activated by Mab to cause ly-sis of LS174T cells. TDA label released to the supernatantwas chelated with europium—the resultant fluorescent che-late EuTDA was detected by time resolved fluorimetry.Complement lysis activity (CMCL Activity) was deter-mined from a standard curve using Mab reference standard.The specific lysis activity was determined by the following:

Specific CMCL activity(U/mg) =CMCL Activity (U/mL)

Mab concentration(mg/mL)

where Mab concentration was determined by nephelometryor the measurement of absorbance at 280 nm.

Oligosaccharide Analysis Using RP-HPLC

0.5 mg of Mab was exchanged into 0.2 M sodium phosphatebuffer, pH 8.0, using Centricon-10 filtration units (Milli-pore, UK) and then incubated at 37°C for 42 h with 20 U ofPeptide-N-glycosidase F (PNGaseF; Boehringer Mann-heim, UK). Released oligosaccharides were purified by pre-cipitation in ice cooled ethanol and dried for PMP (1-phenyl-3-methyl-2-pyrazolin-5-one; Sigma, UK) labelingas previously described (Ashton et al., 1995). Excess re-agent was removed by overnight dialysis against de-ionisedwater using 1000 Da Spectropor dialysis tubing (Pierce,UK). Purified PMP-labeled oligosaccharides were dried un-der nitrogen and reconstituted in de-ionised water forRP-HPLC. RP-HPLC was carried out using a PhaseSepSpherisorb 3ODS2 analytical column (150 mm × 4.6 mmID; Deeside, UK) with detection at 245 nm using an LDCSpectro Minotor D UV detector (Stone, UK). Mobile phasewas delivered using a Spectra Physics (San Jose, USA)ternary gradient pump at room temperature with a flow rateof 0.7 mL/min. The mobile phase consisted of a mixture of0.1 M sodium phosphate, pH 7.0 (Mobile Phase A) andacetonitrile (Mobile Phase B). The required gradient wasdelivered as follows: t4 0 min %A 4 88, %B4 12; t 416 min %A 4 88, %B4 12; t 4 40 min %A 4 84, %B4 16; t 4 44 min %A4 84, %B4 16. The column wasregenerated by elution with a 50:50 mixture of MobilePhases A and B for 6 min before initial conditions wereresumed. The column was equilibrated for 30 min prior tosubsequent injections. Injections were carried out using anSP8880 autosampler (Spectra-Physics, San Jose, USA). Ahuman IgG oligosaccharide library (Oxford GlycoSciences,


UK) was used as an analytical standard in the study—sample peaks were identified by comparison to elutiontimes of the set of standards. Individual peaks in the RP-HPLC chromatogram were collected and purified by dialy-sis against de-ionised water as described above to permitfurther confirmation of identity by assignment of mass byMALDI-TOF (matrix assisted laser desorption/ionisation—time of flight) mass spectrometry.

Oligosaccharide Analysis Using MALDI-TOFMass Spectrometry

Samples for MALDI-TOF analysis were prepared by mix-ing lyophilised peak fractions with 10 mL of 10 mg/mL2,5-di-hydroxybenzoic acid (2,5-di-hydroxybenzoic acidwas solubilised in 10% v/v ethanol/water). A volume of 1mL of the co-crystallisation mixture was then spotted ontoa stainless steel platen and dried at room temperature in thedark. Oligosaccharide analyses were carried out on a Dy-namo mass spectrometer (ThermoBioanalysis, UK) withsample ionisation induced using a nitrogen laser with awavelength set at 337 nm. An asialylated, tetra-antennaryoligosaccharide of known molecular weight was PMP-labeled as described above for use as an internal standard.

RESULTS

Effect of Process Control Parameters on CellCulture Performance

Cell growth and antibody production profiles are shown fortwo representative fed-batch cultures of the total of sixteenof this study, Figure 3. These fed-batch cultures, 1A & 4C,used cell inocula of early (22 d, 11 generations) and late (88d, 60 generations) in vitro age respectively. The measures ofculture performance are summarised for these particularcultures in Table II. Cells in culture 1A proliferated rapidlyto maximum cell density (mave4 0.44 d−1) and entered thedeath phase at day 5. In contrast, culture 4C performedpoorly with a significantly reduced growth rate of 0.28 d−1

and an average specific antibody productivity of 6.1mgMab/106 cells/d. In general, measured values for the se-lected culture responses throughout this study ranged from0.22–0.44 d−1 (mave), 5.7–19.5 × 106 cells-d/mL (IVC), 6.4–32 mg Mab/106 cells/d (qp) and 37–542mg Mab/mL atculture harvest.

Statistical analysis by analysis of variance (ANOVA) ofall data gathered from the study allowed an assessment ofthe effects of the process control parameters at either high orlow outer limits on fed-batch culture responses such as av-erage specific growth rate and specific antibody productiv-ity. Each set of culture response data was modelled with amathematical function indicating the dependence of the cul-ture response on the various control parameters. TheANOVA statistic, the F-value or F-ratio, indicates the ratioof the variability in a response caused by changing a controlparameter, and the variability caused by random error alone.

In this type of design, large F-values (>4) indicate the varia-tion explained by the model is greater than would be ex-pected by chance. The statistical models relating cultureresponses to control parameter variation showed highly sig-nificant outcomes (F-value >4, p < 0.01)—some examples

Table II. Response variables of fed-batch cultures 1A and 4C.

Culture response Culture 1A Culutre 4C

IVC (cells–day/mL) 16.7 × 106 5.7 × 106

mave (d−1) 0.44 0.28qp (mg Mab/106 cells/d) 26.1 6.4Mab titre at harvest (mg/mL) 498 37.2

Culture conditions are described in the caption to Figure 3. IVC4

integral of viable cells,mave4 average specific growth rate, qp 4 averagespecific antibody productivity.

Figure 3. Myeloma cell growth and antibody production in two fed-batchcultures of the study operated under different control conditions. (a) Cul-ture 1A conditions: cells aged 22 d (11 generations) post-revival, pH 7.1,temperature4 38°C, seeding density4 0.43 × 106 cells/mL, feed regimeA, DOT 4 30%. (b) Culture 4C conditions: cells aged 88 d (60 genera-tions) post-revival, pH 7.1, temperature4 34°C, seeding density4 0.27× 106 cells/mL, feed regime B, DOT4 5%.


in the form of 3D plots are shown in Figure 4. The integralof viable cells is dependent on both temperature and cultureseeding density while only temperature had a significanteffect on the specific antibody productivity, Figure 4(a,b).Temperature and seeding density had most significant ef-fects on antibody titre at harvest while all other controlparameters (e.g. DOT in example shown) at either high orlow limits had no effect, Figure 4(c). At the higher limits oftemperature and seeding density, harvest titres were at theirmaximum level—this dependency of Mab titre on these twoparticular control parameters is not surprising as accumu-lated antibody concentration is a function of both the IVC

and qp (Mab titre at harvest4 IVC × qp). A two-factorinteraction effect between dissolved oxygen tension andseeding density on the average specific growth rate wasnoted, Figure 4(d). The explanation for this interaction ef-fect is not entirely clear to us. This culture response wasadditionally greatly influenced by temperature. For ex-ample, at a temperature of 38°C, DOT4 5% and a seedingdensity of 0.27 × 106 cells, the average specific growth ratewas 0.41 d−1 (data shown in Figure 4(d)). However, underthese conditions of dissolved oxygen tension and seedingdensity and at a temperature of 34°C, the model returns asignificantly reduced average specific growth rate of 0.31d−1. Feed regime had no significant impact on any cultureresponse. The effect of this categorical parameter on cultureresponses cannot be presented in a 3D summary plot. In-stead, an effect graph may be used to demonstrate the lackof an impact of feed regime on the magnitude of cultureresponses, such as for example specific antibody productiv-ity in Figure 5. Table III summarises the impact of all con-trol parameters on culture responses.

A gradual decrease in specific antibody productivity wasobserved in the stock 7 L fermenter batch culture used forinoculum supply to each experimental block of fed-batchcultures, Figure 6. However, based on statistical analysis ofdata, no cell-age-related reduction in specific antibody pro-ductivity was observed in fed-batch cultures of any block ofcultures of this study. As much potential block-to-blockvariation as possible was eliminated from the experiment byusing a single experimenter, similar batches of materials,similar equipment etc. for all four blocks of cultures. Toeliminate any possible cell age effect, a fresh revival fromthe WCB would have been made for each block of cultures.However, this was not done for two reasons. Firstly, due tothe experimental approach mentioned above, only cell-ageremained as a possible contributor to a block or run number

Figure 4. Variation of the integral of viable cells (a), the specific anti-body productivity (b), the antibody (Mab) titre at culture harvest (c) and theaverage specific growth rate (d) as a function of various myeloma fed-batch culture control parameters.

Figure 5. Effect of early or late feed regimes on specific antibody pro-ductivity. This minor effect is not significant. The details of the feedregimes are described in the Materials & Methods section.


effect on cell culture responses, and could thus be identified.The statistical software used allows for the analysis of ablock effect (or effect of run number) on any of the mea-sured responses of a blocked factorial experiment, by com-parison of the dimensionless statistic, the studentised re-sidual. The residual is the difference between the actualvalue of the response observed in the experiment (in thisexample specific antibody productivity) and the predictedvalue for the response by the experimental model. The stu-dentised residual (the residual divided by the estimated stan-dard deviation of the residual) versus run number showsrandom scatter indicating no effect of cell age on specificantibody productivity from block to block of the factorialexperiment, Figure 7. To further substantiate this point withmeasured data; myeloma cells were harvested from thestock 7 L fermenter at 88 days post-revival to seed the

fourth and final block of four fed-batch cultures. Whilespecific antibody productivity was 13.3mg Mab/106 cells/dfor the passage prior to harvest of the inoculum, fed-batchcultures 4A and 4D had specific antibody productivities of24.5 and 25.4mg Mab/106 cells/d respectively i.e. at levelscomparable to that of cultures using cells of early in vitroage (see data for culture 1A, Table II). Secondly, the ap-proach was designed to mimic the large-scale processscheme, and verify an in vitro cell age that would encom-pass the cell lifetime in culture at large-scale. In the large-scale process, a single revived vial from the WCB served tosupply cells for more than one production batch. This wasachieved by maintaining the cells in stock bioreactors pre-ceding the production bioreactors in a manner akin to theoperation of the 7 L bioreactor described here.

Although it may appear there is conflict between dimin-ishing productivity with cell age in the stock 7 L bioreactorand the persistently high productivity in the late cultures ofthe factorial experiment, it must be noted that there is afundamental difference between this stock culture (batch)and the cultures of the factorial experiment (fed-batch). Theimplication here is that nutrient feeding restores productiv-ity to the cell line. Other workers have demonstrated theinfluence of medium composition on stability of proteinexpression, and the potential of medium re-feeding or othertreatments to enhance cell-productivity of recombinant pro-tein. For example, Downham et al. (1996) demonstrated thata single re-feed of batch cultures of GS-NS0 cells withglutamate led to an increase in specific antibody productiv-ity (18.9 mg/106 cells/d) compared to un-fed batch controls(12.7mg/106 cells/d)—no data on the effect of this feed oncell growth and/or survival was presented. A hybridomamaintained stable antibody productivity in medium contain-

Figure 6. Specific antibody productivity of GS-NS0 myeloma cells in 7L fermenter batch culture as a function of cell age. The culture was main-tained by passage in fresh growth medium to a target cell density of 0.3 ×106 cells/mL every 72 h. Specific antibody productivity was estimated foreach passage using the following equation: qp 4 (AT2 − AT1).2/(V1 + V2).3where AT1 and AT2 are starting and final concentrations of antibody(mg/mL) respectively and V1 and V2 are starting and final cell densities(cells/mL) respectively for the 72 h (3 d) sub-culture period.

Table III. Summary of the effects of the investigated control parameterson culture responses.

Culture response pH Temperature DOTFeed

regimeSeedingdensity

IVC − + − − +mave − + +/− − +/−qp − + − − −Mab titre at harvest − + − − +

Key to symbols: +, a significant effect wherein the control parameter isincluded in the model as a single parameter effect; +/−, a significant effectwherein the control parameter is included in the model as an interactioneffect with another single parameter (two factor interaction); −, no effect,the parameter is not included in the model. IVC4 integral of viable cells,mave 4 average specific growth rate, qp 4 average specific antibodyproductivity.

Figure 7. Plot of studentized residuals vs. run order for the culture re-sponse, specific antibody productivity. Runs 1–4 were in block 1, runs 5–8were in block 2 etc. Random scatter of the data indicates the lack of aneffect of run number on the culture response.


ing 5% v/v serum but lost production at 1.5% v/v serum(Ozturk and Palsson, 1990), while a recombinant NS0 my-eloma clone expressing a humanised monoclonal antibodyshowed marked differences in long term stability of expres-sion depending on the medium used for its cultivation (Cas-tillo et al., 1999). The specific productivity of a recombinantCHO cell line diminished over forty passages in cultureeven though the integrated gene copy number remainedconstant, and the productivity was restored by sodium bu-tyrate treatment of the cells (Rasmussen et al., 1998).

This phenomenon of diminished productivity with cellage has been observed at all investigated larger scales (80 L& 8000 L) with this particular cell line—data not presented.Our studies have shown that this decrease in productivity inbatch culture with cell age is not due to a lack of clonalityof the WCB. Work is ongoing to determine whether thisoften-observed drop in qp with GS-NS0 cell age is attribut-able to genetic instability (loss of genetic elements encodingMab heavy and/or light chain sequences) or altered expres-sion due to changes at the transcriptional or translationallevel.

Analytical Characterisation ofMonoclonal Antibody

Monoclonal antibody harvested from each of the fed-batchcultures of the study was characterised using SDS-PAGE,IEF, SEC, binding assay and immunoreactivity (CMCL)assay. Six samples selected at random were subjected toglycosylation analysis to further characterise the Mab to itsindividual glycoforms. SDS-PAGE allowed characterisa-tion of Mab by molecular weight of the complete moleculeand the heavy and light chains of the reduced Mab. Elec-trophoretograms of all samples of the study showed bandingpatterns of both the non-reduced and reduced samples to beconsistent with the reference Mab—typical gels for samples1A and 4C are shown in Figure 8. The banding additional tothat of the complete molecule and the heavy and light chainbands give an indication of the purity of the IgG1 prepara-tion. These bands, indicating fragmentation or aggregates ofMab, are barely discernible, if at all, when similar gels arerun using the less sensitive coomassie blue staining method(data not shown). The amounts of aggregated and low mo-lecular weight species in the Mab preparations were quan-tified by size exclusion chromatography. The test samplesconformed to the Mab reference standard specification ofpurity of $95% monomer—both samples shown (1A & 4C)and all others of the study had a purity >99%, Figure 9. Theinvestigation of the ionic charge heterogeneity of Mab pro-duced in all fed-batch cultures of the study showed in everycase, seven distinct isoforms of Mab with isoelectric pointsacross the pI range of 7.40–8.15, Figure 10. Functional as-say data demonstrate that the affinity of Mab for its EGP-40antigen, and its ability to effect the lysis of target cells in thepresence of complement, were comparable to the referencestandard specification for all cases of Mab produced underthe varying combinations of high or low process control

parameters—binding assay and CMCL data are shown forthe representative samples 1A and 4C, Table IV.

The RP-HPLC map of enzymatically-released glycansfrom Mab reference standard is shown in Figure 11. The

Figure 9. Size exclusion HPLC chromatograms of Mab produced infed-batch cultures 1A & 4C. The test articles eluted at 9.78 min (1A) and9.81 min (4C). Mab reference standard eluted at 9.77 min and 9.83 min ascontrols in these runs. Culture conditions are described in the caption toFigure 3.

Figure 8. Silver stained SDS-PAGE gels of Mab produced in fed-batchcultures 1A (a) & 4C (b). M1 and M2 are high and low molecular weightmarkers respectively. Ref is the Mab reference standard. The label ‘Mab’indicates the non-reduced, intact IgG1 band while H & L show the heavyand light chain bands respectively. Ther in the sample notation denotes adithiothreitol-reduced sample. Culture conditions are described in the cap-tion to Figure 3.


profile obtained shows the oligosaccharides expressed to behighly fucosylated with agalactosylated and monogalacto-sylated (peaks 3 and 4 respectively) structures predominat-ing. Other glycans identified include digalactosylated struc-tures with and without bisecting N-acetyl-glucosamine(peaks 1 and 2 respectively) in addition to a small amount ofagalactosylated, non-fucosylated glycans (peak 5). Theseglycans are all N-linked structures located at Asn-297—previous studies have shown that Mab is not glycosylated inthe Fab region (data not shown). Test sample maps (cultures1A and 4C) are practically identical to each other and that ofthe Mab reference standard, Figure 12. The relative propor-tions of the various oligosaccharide structures for each ofthese samples are tabulated for comparative purposes inTable V. Of the 6 samples subjected to glycosylation analy-sis, all showed glycoform distributions similar to that of theMab reference standard. Two (including 1A) of these Mabsamples were produced in fed-batch cultures using my-eloma cells of early in vitro age (22 d, 11 generations) and2 (including 4C) produced using cells of late in vitro age(88.5 d, 60 generations). Two Mab samples produced infed-batch cultures using myeloma cells of mid- in vitro age(64.5 d) showed a slight shift in relative proportions ofoligosaccharide structure from peak 4 (∼5% decrease) to

peaks 2 & 3 (∼2.5–5% increase) compared to Mab referencestandard data (shown in Table V). These minor shifts werenot deemed significant.

DISCUSSION

It is now well established that the titres and quality of gly-coproteins produced in cell culture may depend on manyfactors involved in their production. It is often at the level ofglycosylation that differences in quality become apparent,although this is not always the case. For example, notabledifferences in the degree of sialylation of recombinant hu-man tissue kallikrein were observed when this protein wasproduced by Chinese hamster ovary (CHO) cells grown onmicrocarriers in batch culture or grown in suspended per-fusion culture (Watson et al., 1994). The glycosylation pat-tern of recombinantg-interferon differed when CHO cellswere cultivated on macroporous microcarriers in a perfusedfluidized-bed reactor or in suspension in a stirred tank re-actor (Goldman et al., 1998), while patterns of oligosaccha-ride heterogeneity of IgG1 depended on whether the pro-ducing cell line, a murine hybridoma, was grown in perfu-sion culture in a stirred tank reactor or a hollow fibre reactor(Marino et al., 1997). The host cell line may also have aprofound effect on the characteristics of a recombinant gly-coprotein product. Glycosylation of CAMPATH-1H IgG1differed markedly when it was produced in CHO, Y0 ratmyeloma and murine NS0 myeloma cells (Lifely et al.,1995). Kopp et al. (1997) showed that the characteristics ofsICAM (soluble intercellular adhesion molecule) varieddrastically depending on whether it was expressed in CHOor NS0 cells—these differences were observable by isoelec-tric focusing and SDS-PAGE analyses in addition to oligo-saccharide analysis using HPAEC.

Under conditions in which certain factors are constant i.e.cell line, growth medium, type of bioreactor and culturemode of operation, other factors such as process controlparameters may impact on protein production levels andquality. In continuous culture of a hybridoma in serum-freemedium, a marked increase in the digalactosylated form ofthe Asn-297 oligosaccharide of the secreted IgG1 was ob-served as the dissolved oxygen concentration was increasedfrom 10% to 100% with respect to saturation with air(Kunkel et al., 1998). Lowering of temperature below physi-ological temperature for mammalian cells in culture usuallyresults in reduced growth rates and diminished specific pro-tein productivity (Barnabe´ and Butler, 1994; Sureshkumarand Mutharasan, 1991). However, the productivity of CHOcells producing recombinant antithrombin III was unaf-fected by a culture temperature step-down from 37°C to33°C (Rossler et al., 1996), while the specific productivitywas increased at the lower temperature when recombinantCHO cells producing secreted alkaline phosphatase (SEAP)were cultivated at 30°C and 37°C (Kauffman et al., 1999).No information on the dependence of recombinant productquality on culture temperature was provided in any of theseinvestigations.

Table IV. Measures of Mab biological activity when produced underdifferent fed-batch culture control conditions.

Mab functionCulture

1ACulture

4C

Mab referencestandard

specification

EGP-40 binding activity (U/mg) 1017 1019 700–1300CMCL (U/mg) 1496 1226 600–1400

Culture conditions are described in the caption to Figure 3. CMCL4

Complement-mediated cell lysis.

Figure 10. IEF gel of Mab produced in fed-batch cultures 1A & 4C.Culture conditions are described in the caption to Figure 3. The arrowindicates the position of a faint band that is clearly visible in all samplepreparations on the original gels.


Culture pH is well known to affect cell growth kineticsand specific productivity of recombinant protein (Hayter etal., 1992; Miller et al., 1988; Ozturk and Palsson, 1991), butalso the quality of mammalian cell-produced recombinantprotein. The glycosylation of recombinant mouse placentallactogen-1 produced in a CHO cell line varied significantlywhen the extracellular pH was maintained above pH 8.2 andbelow pH 6.9 (Borys et al., 1993). Glycosylation of thismolecule was subsequently shown to be dependent on aninteraction between pH and ammonium ion concentration(Borys et al., 1994). The degree of polysialylation of neuralcell adhesion molecule was dependent on CHO cell culturepH, and the pH effect on protein quality was additionallydependent on culture pCO2 (Zanghi et al., 1999). Cultureharvest time may also cause variations in glycosylated pro-tein quality. A shift towards high-mannose-type N-glyco-sylated IgG1 was observed as a fed-batch culture of GS-NS0 myeloma cells was extended up to 22 d (Robinson etal., 1994).

Although many parameters in a typical batch cell cultureprocess may be controlled, it is still very much a dynamicsystem in which primary nutrients such as glucose, gluta-mine and amino acids become depleted and metabolic wasteproducts such as lactate and ammonia accumulate. Fed-batch culture is carried out to ensure essential nutrients aresupplied at optimal concentrations or in excess for the du-ration of the culture but does not allow for removal ofundesirable by-products of metabolism. In addition, in-creases in osmolality during fed-batch culture are commonif carried out using concentrated feed media. These factorsmay also impact on protein quality—as part of their study,Schill and co-workers (1998) showed daily variation in thepopulation of glycoforms of a recombinant protein during

fed-batch culture of a CHO cell line. Gawlitzek et al. (1998)showed that the presence or absence of glutamine and am-monium ion had an impact on the quality of a mutant variantinterleukin-2 produced by BHK-21 cells in continuous cul-ture.

These cited examples illustrate some of the many factorsto consider for the production of appropriate quality glyco-protein in a particular cell culture manufacturing process.The purpose of this protocol was not to identify the ex-tremes of process control parameters or other culture con-ditions that impact on the quality of Mab when produced inthis defined fed-batch cell culture system. Our goal was toverify that process performance was unchanged and Mabcharacteristics remained unaltered when produced withinhigh and low levels of critical control parameters routinelycontrolled during production cell culture. This remains thegoal of any cell culture process validation exercise. Drawingfrom process knowledge and early development experience,a half-fractional factorial experimental design was con-structed to generate random combinations at their outer lim-its of five process control parameters that could most likelyimpact on product quality. Certain factors known to affectglycoprotein quality were considered, but deemed of noconcern in this experimental protocol. For instance, glucosestarvation never occurs in this culture system due to peri-odic feeding of glucose-containing feed medium, and am-monium ion does not accumulate in the culture due to thenature of the glutamine synthetase expression system whichpromotes the scavenging of ammonia for the synthesis ofglutamine from glutamate. While osmolality typically var-ied between 300–400 mOsm/Kg H2O and pCO2 between20–140 mmHg during this fed-batch culture under optimalconditions, we considered these variables within ranges not

Figure 11. The RP-HPLC map obtained for the oligosaccharides released from Mab reference standard and subsequently labeled with PMP. Individualpeaks were isolated for further confirmation of identity by MALDI-TOF mass spectrometry analysis (data not shown).


likely to affect product quality based on our in-house expe-rience with this, and other, GS-NS0 cell lines. Agitation rateand culture power input were previously determined non-critical across a wide range of values. The resultant analysisof data enabled the quantification of all the main factoreffects (effects of single parameters) on culture responsessuch as growth rate and productivity. The effects of two-factor interactions, for example the dissolved oxygen/seeding density interaction effect on culture average spe-cific growth rate (mave) were also identifiable. This wouldnot have been possible if the more traditional approach ofvarying one-parameter at a time while controlling all othersat a constant level was utilised. In addition, this latter ap-proach to executing process development and validationprotocols is much more intensive in labour and time thanstatistical DOE. This powerful experimental tool has thus

permitted the definition of a culture operating space or re-gion in which all fed-batch culture control parameters mayvary to their extremes, either singly or all together, with nodeleterious effect on product quality. This operating spaceexists at all points within the validated proven acceptableranges of pH 7.1–7.5, DOT 5–30%, temperature 34–38°C,seeding density 0.27–0.43 × 106 cells/mL, culture feedingtimes at 48 ± 4 h, 96 ± 4 h, 144 ± 4 h andculture harvestat 168 ± 4 h. It isworth noting that while a temperature of34°C in combination with all levels of other parameters didnot have an adverse effect on product quality, growth ratesand harvest titres at this lower limit of temperature werereduced to levels considered too low for the economicalcommercial manufacture of a monoclonal antibody. Thestatistical model suggests that a lower limit of temperatureof 35.2°C would result in increases in culture performanceto the minimum acceptable levels of growth rate and pro-ductivity based on in-house acceptance criteria. Hence, atemperature control parameter range for manufacturingwould ordinarily be selected such that its lower limit lay asafe distance from the edge of process failure of this criticalparameter (34°C in this study). This is the simple conceptunderpinning the strategy of “engineering” critical param-eters out of the process as recently described by Seely et al.(1999). In this article, an ion exchange chromatography op-eration in the purification process of a recombinant proteinwas described in which process failure occurred due to poorbinding of the protein to the resin during the resin-loadingstep. The failure was attributed to the pH of the load solu-tion which was initially deemed to be pH 6.20 but wasdetermined on re-measurement post-processing to be pH6.32. Subsequent investigative lab-scale runs confirmed thatpH 6.20 and 6.34 resulted in successful and failed chroma-tography runs respectively. Through introduction of mea-sures to prevent operator error/equipment failure, and set-ting the upper limit of pH for the load solution at pH 6.1, theload solution pH was transformed from a critical variable toa non-critical one. It is true that if the upper and lower limitsof the proven acceptable range for a particular control pa-rameter are sited sufficiently distant from the upper andlower limits of the edges of failure, there is greater assur-ance that fluctuations in the control parameter will not im-pact on process performance, product quality or both. How-ever, this author does not concur with these author’s (Seeleyet al., 1999) opinion that a process may be engineered tocreate a non-critical process control parameter from a criti-cal one. According to our terminology, a critical processcontrol parameter will always remain thus, regardless ofwhere its operating set-point and proven acceptable rangeare set relative to its edges of failure, since failure of controlof this parameter will inevitably result in process and/orproduct failure.

The described validation exercise would not constitute acomplete validation program for a bioprocess. Following astudy such as this, an appropriately protocoled performancequalification (PQ) program would be carried out to demon-strate that the bioprocess could yield safe, efficacious prod-

Table V. The relative % peak areas for the analysis of oligosaccharidesenzymatically released using PNGaseF from Mab samples harvested fromrepresentative fed-batch cultures of the study.

Culture reference Peak 1 Peak 2 Peak 3 Peak 4 Peak 5

Mab Reference Standard 0.3 3.6 42.8 45.8 1.51A 1.5 5.6 46.7 43.9 1.14C 1.0 5.8 48.9 43.2 1.1

The identities of each peak (1–5) are detailed in Figure 11. Cultureconditions (1A & 4C) are described in the caption to Figure 3.

Figure 12. The RP-HPLC oligosaccharide map obtained for representa-tive fed-batch culture samples 1A and 4C. The identity of each peak isdetailed in Figure 11. Culture conditions are described in the caption toFigure 3.


uct of specified quality in consecutive (usually three) GMPproduction batches at large-scale. Extensive testing andcharacterisation of purified product would accompany thiscampaign. A demonstration of the consistency and repro-ducibility of the process performance (cell growth kinetics,productivity etc.) is also an essential element of the PQexercise. In our hands, these PQ batches would be operatedat control parameter optima. Although some fluctuation ofcontrol parameters about the set-points would be normal atlarge scale, they would not typically stray to their upper orlower limits during the PQ program. Hence, to validate thecontrol ranges, or identify acceptable “upper and lower pro-cessing limits” as prescribed by CDER’s Guideline on Gen-eral Principles of Process Validation (1987), we carry outvalidation work as outlined in this article. In conclusion, weuse studies such as that described here as part of an exten-sive validation program to provide confidence that lab-scaleand pilot-scale cell culture processes will transfer to thelarge-scale manufacturing environment with an appropriatedegree of robustness for producing biologicals of acceptablequantity, safety and quality.

The author gratefully acknowledges the contribution of Cather-ine Bentley and Fiona Montague (Biopharmaceutical ProcessSciences, GlaxoWellcome Research and Development) for theprovision, and discussion, of early process development data.Richard Lyons (Statistical Services, GlaxoWellcome Researchand Development) advised on the statistical experimental designelement of this work.

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