U2P3_mean-value-theorem-antiderivatives-and-differential-equat

Effect of Antibody Solution Conditions on Filter Performance forVirus Removal Filter Planova TM20NTomoko Hongo-Hirasaki,Masayasu Komuro,and Shoichi IdeTechnology Development,Planova Division,Asahi Kasei Medical Co.Ltd.,2700Asahimachi6-chome,Nobeoka,Miyazaki882-0847,JapanDOI10.1002/btpr.415Published online March2,2010in Wiley Online Library().We investigated the effect of antibody solution conditions(ionic strength,pH,IgG concen-tration,buffer composition,and aggregate level(dimer content))onfilter performance for avirus removalfiltration process using the Planova TM20N,a virus removalfilter.Ionicstrength and pH affected thefilterflux.A consistent highflux was maintained at an ionicstrength greater than10mM and at pH4–8under a typical buffer composition(sodiumchloride,citrate,acetate,and phosphate).Optimum IgG concentration was10–20mg/mLallowing for high throughput(kg/m2of IgG).Dimer content negligibly affected theflux level.Under high throughput conditions,virus spiking did not affectflux whereas a parvovirus log-arithmic reduction value greater than5was maintained.From the results of zeta potentialanalyses for IgG and the membrane,we considered that electrostatic interactions betweenantibodies and the membrane affectfilter performance(flux level and throughput).Theseresults indicate that the Planova TM20Nfilter is applicable for a wide range of solutionconditions typically used in antibody processing.V C2010American Institute of ChemicalEngineers Biotechnol.Prog.,26:1080–1087,2010Keywords:virus removalfilter,antibody,electrostatic interaction,filter performance,solution conditionsIntroductionIn downstream antibody production,virus removalfiltra-tion operations are conducted under a wide range of solution conditions.These differences are the result of product/pro-cess optimization.Typical virus removalfiltration steps are conducted following chromatography,and these result in var-iations in solution composition.1–6Figure1shows the typical virus removalfiltration step in downstream antibody produc-tion.These variations in solution include antibody concentra-tion,ionic strength,pH,buffer composition,and aggregate level.These variations in virusfiltration load material may therefore affectfilter performance.Mouse parvovirus contamination in CHO-derived proc-esses has been reported previously.7Authorities have indi-cated the necessity for virus removal or inactivation in the mammalian cell culture production process.8Therefore, small virusfilters targeted for parvovirus removal have been applied to the monoclonal antibody(mAb)process.9 Although the effectiveness of parvovirus removal in antibody solution by small virus removalfilter have been reported,9,10 in the view offilterability,the relationship betweenfilter per-formance and solution conditions of antibody has not been clarified for small virus removalfilter.Forfiltration using an ultrafiltration membrane,the effects of solution pH onfiltration has been studied.11,12In these reports,fluxes showed minimum values at a pH equal to the isoelectric point(pI)of the protein and increased with low and high pH values.While examining the relationship between membrane elec-trical charge and ionic strength,it was found that the perme-ability of cytochrome C through a charge-modified UF membrane(cutoff MW:30kD)increased with increasing ionic strength.13This dependency was observed more clearly in a membrane with a high electrical charge.Thus,for the ultrafiltrationfield,it has been considered that the physico-chemical property of the membrane and protein solutions such as electrostatic interaction is an important factor for determining the performance offiltration in addition to size-based separation.However,for a small virus removalfilter,thefilter per-formance for IgG solution has not been investigated system-atically from the viewpoint of the effect of solution conditions.This article focuses on evaluating thefilter performance (flux,flux decay,and throughput)of the Planova TM20N for IgG solution and clarifying the relationship betweenfilter performance for IgG solution and solution conditions such as pH,ionic strength,IgG concentration,and aggregate level. To clarify the electrostatic interaction,the zeta potential was measured for IgG and membranes under various values of pH and ionic strength.We also demonstrated the optimal so-lution conditions for a small virus removalfiltration process. The IgGfiltration mechanism is discussed with respect to surface interaction between IgG and the membrane of the vi-rusfilter.Correspondence concerning this article should be addressed to T.Hongo-Hirasaki at hongo.tb@om.asahi-kasei.co.jp.1080V C2010American Institute of Chemical EngineersMaterials and MethodsMaterialsSmall Virus Filter.Planova TM20N(0.001m2)(Asahi Kasei Medical Co.Ltd.,Japan)was used as a small virus re-movalfilter.It is composed of regenerated cellulose hollow fiber membrane.14IgG Solution.Human IgG(Venoglobulin V R-IH)(Mitsu-bishi Tanabe Pharma Corporation,Japan)was used as a model IgG.This IgG is a polyclonal antibody,and therefore the range of pI was broad pH6.8–10as determined by iso-electric focusing analysis using precast polyacrylamide gel (IEF–PAGE mini)(TEFCO Co.,Japan)with ampholine(pH 3-10).The original solution contained50mg/mL IgG and50 mg/mL sorbitol as a stabilizer,i.e.,each diluted IgG solution also contained a corresponding sorbitol concentration.Sorbi-tol,a kind of sugar alcohol,is known to stabilize the disper-sion of protein due to hydration on protein surface.Sorbitol itself below50mg/mL(maximum amount in this model IgG)without IgG little affected thefilterflux(data not shown).To investigate the effect of ionic strength,10mg/mL IgG solutions were prepared in0,0.1,1,10,50,100,300,500, and1000mM NaCl solutions.To investigate the effect of IgG concentration,1,5,10, 20,30,and50mg/mL IgG solutions were prepared in0or 100mM NaCl solutions.The pH of10mg/mL IgG in100mM NaCl solution was adjusted to a pH ranging from3to9using1M HCl and 1M NaOH to investigate its effect.For a typical buffer case,citrate buffer(sodium citrate) (pH3.3–5.4),acetate buffer(acetates,sodium acetate)(pH 3.6–5.6),and phosphate buffer(pH5.7–7.9)were used and prepared at an ionic strength of0.7–463mM in10mg/mL IgG solution.Virus.Porcine parvoviruses(PPV)(90HS strain,Japa-nese Association of Veterinary Biologics,Japan)were propa-gated in PK-13(ATCC Number CRL-6489)cells at37 C. The growth medium was Dulbecco’s modified Eagle’s me-dium with D-glucose1000mg/L and L-Gln584mg/L (Nikken Seibutsu Igaku Laboratory,Japan)containing3% fetal bovine serum(FBS).Serum-free PPV was obtained from the supernatant after exchanging the medium without FBS before a cytopathic effect appeared in the host cell after virus inoculation.The supernatant was collected and centri-fuged at4 C,3000rpm for20min to pellet down the cell fraction.The supernatant was passed through a0.45-l mfilter (membrane diameter50mm;SFCA Filter Nalgene Nunc International).The concentration of the serum-free PPV stock solution is expressed in terms of50%tissue culture in-fectious dose(TCID50)and was about108.5TCID50/mL. MethodsFiltration Performance.IgG solutions werefiltered through a small virusfilter Planova TM20N under a dead-endfil-tration mode at a constant pressure of78.4kPa(11.4Psi)at 25 C.Flux,filtration volume,and throughput(IgG kg/m2)were evaluated.V max as the indicator offlux decay was calculated fromflux versusfiltration time9,15and expressed as Eq.1.t=V¼ð1=V maxÞtþ1=Q0(1) where t¼filtration time,V¼cumulativefiltrate volume at time t,V max¼estimated maximum volume that can befil-tered,Q0¼initialflux at start offiltration(t¼0).High-Performance Liquid Chromatography–Size Exclusion Chromatography(HPLC–SEC)Analysis for Model IgG.The aggregate level of IgG was measured by HPLC–SEC using Prominence HPLC(Shimadzu,Japan)and a TSK-Gel G3000SW XL column(Tosho Bioscience,Japan). The mobile phase was300mM phosphate at pH 6.9, 200mM arginine-HCl,and100mM NaCl.16Theflow rate was1mL/min at25 C.Zeta Potential Analysis for the Model IgG and Virus Removal Membrane.The surface charge on IgG under the various concentrations of NaCl(1–100mM)and various val-ues of pH(4,5,6,7,and8)was determined using a zeta potential analyzer ELS-Z(Otsuka Electronics Co.Ltd.,Ja-pan).Zeta potential analysis is based on electrophoretic light scattering.17The zeta potential of IgG was calculated from electrophoretic mobilities.Zeta potential analysis of IgG was performed using the pH Titration System ELSZ-PT(Otsuka Electronics).The zeta potential of the membrane surface was measured with a cell for a solid-plate sample using a monitor particle(Otsuka Electronics).17Instead of a hollowfiber-type membrane,we prepared aflat-sheet-type of regenerated cellulose mem-brane18to measure the zeta potential on the surface of the cellulose membrane.Virus Spiking Studies.The serum-free PPV prepared in above section was spiked into1,5,10,20,30,50mg/mL IgG in100mM NaCl solution(pH5)at0.5%vol/vol.Vi-rus-spiked IgG solutions werefiltered through Planova TM 20N under a dead-endfiltration mode at a constantpressure Figure1.The typical virusfiltration step in downstream antibody production.of 78.4kPa (11.4Psi)at 25 C.All feed samples were held for a processing time.The PPV titer of feed solution was ca.6.0–6.5log 10(TCID 50/mL).Quantification of Virus Concentration.We determined the viral concentration in the sample before and after filtra-tion by the TCID 50method using PK-13cells.After holding,samples of the feed solution were also determined.Ten-fold serial dilution of samples was tested in cell culture plates.TCID 50calculation was according to the Reed &Muench method.19Virus titers were expressed as TCID 50/mL.The detection limit of this method in this experiment is 0.5log 10(TCID 50/mL).After holding,the decrease in the activity of the feed sample was negligible.Retention values are calcu-lated as the logarithmic reduction value (LRV)from Eq.2.LRV ¼Àlog 10C f =C o(2)where Co and Cf are the concentrations of virus in the feed solution and filtrate,respectively.Results and DiscussionEffect of ionic strength on filter performancesTo simplify the effect of ionic strength,ionic strength was controlled with NaCl due to equivalent to molar concentra-tion of salt.Figure 2a shows the flux profiles with the Plano-va TM 20N virus removal filter obtained over 0to 1000mM NaCl.At each NaCl concentration,an almost constant flux was sustained over the filtration volume.A little flux decay was observed at [300mM NaCl.There appeared to be a de-pendence of steady-state flux on NaCl concentration (Figure 2b).Flux level increased with NaCl concentration and showed maximum at around 150mM NaCl.Addition of [10mM NaCl enhanced the flux about 1.5–2times higher than at lower NaCl concentrations (\1mM).Figure 3shows the V max and dimer content of IgG solution as a function of NaCl concentrations from 0to 1000mM.In this experiment,we could not observe multimers by HPLC–SEC analysis.The dimer content increased with NaCl concentration,whereas V max decreased with it.Without IgG,flux with Pla-nova TM 20N was not influenced by NaCl concentration.We found that the flux level for IgG filtration was controlled with NaCl concentration,and slightly affected by the dimer content,whereas flux decay expressed as V max was consider-ably affected by it.From this result,we consider that surface interaction may influence the antibody filtration performance.It is well known that hydrophobic interaction increase with increasing NaCl concentration.Under these circumstances,it is consid-ered that dimer formation of IgG accelerated due to the hydrophobic interaction between IgG itself.On the other hand,the virus removal filter Planova TM 20N is composed of regenerated cellulose hollow fiber membrane 14and has hydrophilic properties.20Therefore,we considered that hydrophobic interaction between IgG and the cellulose mem-brane was minimized with respect to NaCl concentration.Another interaction (electrostatic interaction)between IgG and the membrane is discussed in a later section.Effect of IgG concentration on filter performances (0and 100mM NaCl)Filter performance for various IgG concentrations was examined at fixed ionic strength of lower (0mM)andFigure 2.Flux profiles for 10mg/mL IgG solution using Plano-va TM 20N at various NaCl concentrations (pH 5.4).(a)Flux versus filtration volume,*:0mM,l :0.1mM,~:1mM,~:10mM,n :100mM,þ:150mM,h :300mM,^:500mM,n :1000mM.(b)Average flux versus NaClconcentration.Figure 3.V max and the dimer content of 10mg/mL IgG solu-tion using Planova TM 20N as a function of NaCl con-centration (pH 5.4).*:dimer content,l :V maxoptimum (100mM)level,which showed high flux.Figure 4shows the average flux with Planova TM 20N obtained over a broad range of IgG concentration from 1to 50mg/mL at 0mM NaCl and 100mM NaCl.The flux of 1–30mg/mL IgG with 100mM NaCl was larger than that with 0mM NaCl.The flux of 50mg/mL IgG with 100mM NaCl was slightly smaller than that with 0mM NaCl.At a high concentration of IgG ([30mg/mL),hydrophobic interaction between IgG itself with NaCl addition may play a role in the decrease of flux level.Figure 5shows the dimer content of various con-centrations of IgG with 0and 100mM NaCl.The dimer content of 1–50mg/mL IgG with 100mM NaCl was higher than that with 0mM NaCl.The dimer content increased with increasing IgG concentration.At 50mg/mL IgG,dimer level slightly decreased in compared with 30mg/mL IgG.This may be because optimum concentration of sorbitol to stabilize IgG is around 50mg/mL.Furthermore,we alsocould not observe multimers by HPLC–SEC analysis.It seems that the dimer content in 1–30mg/mL IgG negligibly affected the flux level except at high IgG concentration ([30mg/mL).Figure 6shows the throughput of IgG for 3-h and 5-h filtrations using Planova TM 20N at 0and 100mM NaCl.The throughput was expressed as the filtered amount of IgG per filter surface area (kg/m 2).The throughput with 100mM NaCl was higher than that with 0mM NaCl.The optimum concentration of IgG for Planova TM 20N to obtain high IgG throughput was 10–20mg/mL under these solution condi-tions.At a high concentration of IgG ([30mg/mL),the IgG throughput at a longer filtration time (5h)at 0mM NaCl showed nearly equal to or slightly higher than the throughput at 100mM NaCl.At ionic strength of 100mM,which showed high flux,we investigated the effect of virus spiking on filter performance as a function of IgG concentration (1–50mg/mL)with spike percentage of 0.5vol %PPV (Figure 7).Flux profiles with virus spiking were similar to that of each control without virus spiking.Virus spiking didn’t affect flux profile for vari-ous IgG concentrations.Under these conditions,Planova TM 20N showed high PPV LRV [5for wide range of IgG con-centration (1–50mg/mL)for 5h filtration (Table 1).PPV LRV was independent of IgG concentration.Effect of pH (100mM NaCl)on filter performance Filter performance was examined as a function of pH fixed at ionic strength of 100mM.Figure 8shows average flux with Planova TM 20N versus pH.At pH 3and pH 9,a remarkable flux decay was observed over filtration volume.At pH 4–8,an almost constant flux,which showed a slightly different level at each pH,was maintained over the filtration volume,similarly shown in Figure 2a.The average flux level of 10mg/mL IgG with Planova TM 20N was highest at pH 4–5.Figure 9shows the V max and the dimer content of IgG so-lution at different pH.At pH 3,IgG multimer (larger than trimer)was detected by HPLC–SEC analysis most likely due to low pH denaturation (data not shown).At pH 4,dimer level was minimum and then increased with pH.At pH 9,although a multimer was not detected byHPLC–SECFigure 4.Average flux for Planova TM 20N as a function ofIgG concentration.*:0mM NaCl concentration (pH 5.4)l :100mM NaCl con-centration (pH5.4)Figure 5.Dimer content as a function of IgG concentration.l :0mM NaCl,*:100mMNaCl.Figure 6.Throughput of IgG for 3h and 5h filtrations usingPlanova TM 20N.l :100mM NaCl,3h,*:100mM NaCl,5h,~:0mM NaCl,3h,~:0mM NaCl,5h.Throughput was calculated as conversion at 98kPa.analysis,denaturation of IgG may occur.We consider that multimers may cause fouling of the Planova TM 20N filter.Detailed analysis for the effect of multimers on IgG filtration will be discussed in the forthcoming article (Hongo-Hirasaki T,Komuro M,Hayashida H,Ide S,in preparation).At pH 4–5,the V max level was highest;however,at pH 7–8(close to the pI of the model IgG),it decreased.The dimer content of IgG increased at the pI.It was considered that hydropho-bic interaction between IgG–IgG increased at the pI region of the model IgG.From these results,except for pH 3and pH 9,dimer content didn’t so impact flux level but cause flux decay.Effect of typical buffer solutions (citrate,acetate,and phosphate)We selected citrate,acetate,and phosphate as typical buffer solution in process.These have different number of ionic valence and dissociation value.Therefore,we expressed the concentration of each buffer composition as ionic strength.Figure 10shows the dependence of average flux on ionic strength for 10mg/mL IgG in typical buffer solutions using Planova TM 20N.The average flux level increased with ionic strength greater than 20mM for citrate,acetate,and phosphate buffers as well as that for NaCl.The average flux level with buffer solutions was 1.5–2.5times higher than that with an ionic strength of 0mM (the flux for 10mg/mL IgG was ca.20L/m 2/h from Figure2a,b).Figure 7.Effect of parvovirus spike on flux profile at varyingIgG concentration using Planova TM 20N.(100mM NaCl,pH 5)*:1mg/mL IgG,without PPV,l :1mg/mL IgG,with PPV,~:5mg/mL IgG,without PPV,~:5mg/mL IgG,with PPV,h :10mg/mL IgG,without PPV,n :10mg/mL IgG,with PPV,^:20mg/mL IgG,without PPV,^:20mg/mL IgG,with PPV,:30mg/mL IgG,without PPV,:30mg/mL IgG,with PPV,:50mg/mL IgG,without PPV,þ:50mg/mL IgG,withPPVFigure 8.Average flux of 10mg/mL IgG in 100mM NaClsolution for Planova TM 20N at pH3–9.Figure 9.V max and the dimer content of 10mg/mL IgG in 100mM NaCl solution as a function of pH.*:dimer content,l :V max .The shaded region is expressed as pI region of this modelIgG.Figure 10.Average flux as a function of ionic strength forcitrate,acetate,and phosphate buffers at various pH (3.3–7.9).*:Citrate,pH ¼3.3,~:Citrate,pH ¼4.3,h :Citrate,pH ¼5.4,l :Acetate,pH ¼3.6,~:Acetate,pH ¼4.8,n :Ace-tate,pH ¼5.6,þ:Phosphate,pH ¼5.7,n :Phosphate,pH¼6.9,^:Phosphate pH ¼7.9.Table 1.Porcine Parvo Virus (PPV)LRV with Planova TM 20N for 5h Filtration at Varying IgG Concentration IgG Concentration (mg/mL)PPV LRV for Filtrate Pool1!5.4210!6.0020!5.7530!5.8650!5.67Addition of buffer component enhanced the flux level for IgG filtration using Planova TM 20N.Zeta potential of IgG and the virus removal membrane To clarify the mechanism of the effect of solution condi-tions (ionic strength and pH)on IgG filtration performances,we focused on the electrostatic interaction between IgG and the membrane to assess a surface charge.Figure 11shows the zeta potential of the model IgG under various conditions of pH (4–8)and NaCl concentration (0–100mM).In 50and 100mM NaCl,we could not deter-mine the zeta potential of IgG at pH 6–8due to aggregation during electrophoresis.The zeta potential of IgG was posi-tively charged at a pH region below the pI of model IgG(6–8).At low NaCl concentration (0–10mM),the positive zeta potential was higher than that at [10mM NaCl.In par-ticular,at pH 4and 0mM NaCl,the zeta potential of IgG was high;ca.þ35mV.The level of positive zeta potential decreased with NaCl concentration and increase in pH.At pH 6–8,near the pI region of IgG,the zeta potential of IgG achieved an almost zero value at [50mM NaCl.In low NaCl concentration at the pI region,IgG was slightly posi-tively charged.In low NaCl concentration,highly positively charged IgG itself was stably dispersed in solutions,causing reduction in aggregate (dimer)formation.Figure 12shows the zeta potential of cellulose membrane under various conditions of pH (4–7)and NaCl concentration (1–100mM).The zeta potential of the surface of the cellu-lose membrane was negative at pH 4–7.The negative zeta potential of the membrane surface decreased with increasing NaCl concentration.The negative zeta potential of the mem-brane surface increased with increasing pH.We consider that the negative charge of regenerated cellulose membrane may correspond to hydroxyl pared with the zeta potential of IgG,the change in the zeta potential of the membrane surface with NaCl concentration more than 5mM was relatively small.From the aforementioned results,we considered the elec-trostatic interaction between 1)IgG and the membrane and 2)IgG and IgG under these experimental conditions.1)IgG and membraneAt conditions of low ionic strength below the pI of the model IgG,highly positively charged IgG may attract to the negatively charged membrane.This increases the resistance of fluid flow inside the narrow pore space of the membrane,reducing the flux level for the filter.Therefore,a high filter performance (flux)was achieved with the appropriate ionic strength ([10mM NaCl)and pH (below the pI of IgG)that corresponded to weaken the attractive interaction between the oppositely charged IgG (positive)and membrane (negative).2)IgG and IgGAt low ionic strength,highly positively charged IgG may repulse between IgG molecules.It is considered thatthisFigure 11.Zeta potential for IgG as a function of pH (4–8)atvarying NaCl concentration (0–100mM).*:0mM NaCl,l :10mM NaCl,~:20mM NaCl,~:50mM NaCl,n :100mMNaClFigure 12.Zeta potential for regenerated cellulose membraneas a function of pH (4–8)at varying with NaCl con-centration (1–100mM).*:1mM NaCl,~:5mM NaCl,h :10mM NaCl,^:100mMNaCl.Figure 13.The flux profile of 10mg/mL IgG in 100mM argi-nine or 100mM NaCl solution for Planova TM 20N (pH 55.4).*:0mM NaCl,l :100mM Arginine.repulsive interaction is reflected in the dispersion stability of IgG in the solution.Effect of additives (amino acids)on IgG filtrationFigure 13shows the flux profile of 30mg/mL IgG with addition of an amino acid (arginine)through Planova TM 20N.In case of a high concentration of IgG (30mg/mL),a saline buffer system showed low flux;however,addition of 100mM arginine improved the flux level 2.5times higher than 100mM NaCl.In addition to arginine,histidine also improved the flux of Planova TM 20N at high concentration of IgG (Hongo T,Komuro M.Jpn Pat.Patent Pending).Fig-ure 14shows the throughput of IgG using Planova TM 20N with arginine addition for 3h and 5h filtrations.In compari-son with 100mM NaCl,the optimum concentration of IgG to obtain maximum throughput of IgG was shifted to a high concentration ([30mg/mL)with the improvement of IgG throughput.Addition of arginine or histidine reduced the sur-face charge of antibody (þ35mV at pH 4and 0mM NaCl to ca.þ15mV at pH 4and 100mM arginine or histidine).On the other hand,arginine and histidine didn’t increase the dimer level compared with NaCl (data not shown).There-fore,we consider that this effect is due to weaken both elec-trostatic interaction between IgG–membrane and hydrophobic interaction between antibodies.Contribution of surface interaction to filter performance Solution conditions (pH and NaCl concentration),which are assumed to be applied in downstream process of anti-body production,affect the flux of IgG for Planova TM 20N filtration.Regenerated cellulose,which is the material of the Planova TM 20N filter,is hydrophilic and was known for its low nonspecific interaction with proteins.20Therefore,we consider that the hydrophobic interaction between IgG and the membrane for the Planova TM 20N filter are negligible.At a pH range of 4–7below the pI region of IgG,the zeta potential of IgG was positive and that of the membrane was negative.Therefore,electrostatic interaction between IgG and the cellulose membrane may cause the resistance of flow inside the pore of the membrane during filtration.This shifts the flux to a low level under low ionic strength.When the ionic strength is high enough (i.e.,[10mM)to shield the electrostatic interactions,the hydrophobic interaction between IgG itself increases at the pI region (the zeta poten-tial of IgG was almost zero).This interaction may reduce the stability of dispersion of IgG in the solution showing an increase in the dimer content.It is considered that when IgG passes though the narrow pore of the membrane,IgG itself may aggregate due to hydrophobic interaction.Therefore,it is considered that under conditions of ionic strength greater than 50mM and/or the pI region,we observed flux decay expressed as the decrease in V max .At a high concentration of IgG ([30mg/mL),the hydrophobic interaction between IgG molecules may affect the filter performances (flux level reduction and flux decay)to a considerable extent.Addition of some amino acids such as arginine and histi-dine improved the flux level for high concentrations of IgG.These amino acids reduced the surface charge of antibody.It is considered that arginine weakens the electrostatic interac-tion between IgG and the cellulose membrane,and the hydrophobic interaction between protein molecules.It was reported that for virus filtration for a mAb solution in the same buffer solution,different types of mAbs showed different filter performances.9This may be due to differences in biophysical properties (surface charge)between them.Our results were obtained from a model IgG (polyclonal human IgG),and therefore the degree of the zeta potential of mAb may be different from our model IgG,and the efficiency of ionic strength and pH on filter performance may appear more drastic.Furthermore,detailed characterization of bio-physical properties (surface charge)for mAbs under various solution compositions will be needed to optimize the virus filtration process for antibody production.ConclusionsFrom above results,we can conclude the IgG filtration phenomena using Planova TM 20N as follows:•The composition of IgG solution (ionic strength and pH)affects the flux of small virus filter Planova TM 20N.•For this model antibody,optimal solution conditions are ionic strength of more than 10mM and pH ¼4–6below pI of this antibody.•The most economical IgG concentration for Planova TM 20N filtration is 10–20mg/mL.•Under high throughput conditions,virus spiking did not affect flux while maintaining parvovirus LRV greater than 5.•Surface interaction influences the filter performance.Electrostatic interaction between IgG and the membrane mainly affect the flux level,and the hydrophobic interaction between IgG–IgG molecules affect the flux decay of small virus filtration for 30mg/mL IgG.•Some additives (e.g.,the amino acids,arginine and histi-dine)improve the flux level for high concentration ([20mg/mL)of IgG filtration.Although the efficiency of ionic strength and pH might differ in degree depending on each monoclonal antibody,basically these phenomena obtained from the model polyclonal IgG will be applicable for actual monoclonal antibodyfiltration.Figure 14.Throughput of IgG in 100mM arginine or 100mMNaCl solution for 3-h and 5-h filtrations using Planova TM 20N (pH 55.4).l :100mM Arginine,3h,*:100mM Arginine,5h,~:0mM NaCl,3h,~:0mM NaCl,5h.Throughput was calculated as conversion at 98kPa.AcknowledgmentsWe acknowledge the useful advice by Mr.Shoichi Nakamura (Otsuka Electronics Co.Ltd.,Japan)regarding zeta potential analysis.And we also thank Ms.Sachiko Goshi and Ms.Akiyo Kajitani for thefiltration experiment in the Planova Division of Asahi Kasei Medical Co.Ltd.,Japan.Literature Cited1.Birch JR,Racher AJ.Antibody production.Adv Drug DelivRev.2006;58:671–685.2.Shukla AA,Hubbard B,Tressel T,Guhan S,Low D.Down-stream processing of monoclonal antibodies-Application of 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Recommendations for Victor 3/X4/X5 plate readersThe Perkin Elmer Victor 3/X4/X5 plate reader with TRF capability allows the use of the MitoXpress Xtra Oxygen Consumption Assay with dual-read TRF lifetime detection using well-wise kinetic measurements of two labels with different TRF lag times. These two TRF intensities allow the ratio-metric calculation of fluorescence lifetimes, representing the level of oxygen in each sample.Perkin Elmer Victor 3/X4/X5 readers must be equipped with suitable filter sets. For optimal performance, we recommend using dual-read TRF lifetime detection, and setting up the measurement conditions in the instrument control software according to the following instructions.1. Create a Protocol in the Perkin Elmer 2030 ExplorerApplication.2. Open the Protocol and Add a TRF Label in theMeasurement Tab.3. Edit the Properties of the Label to set the parameters asdescribed in the following instrument setup table.Please refer to the Perkin Elmer instrument software user manual for further information.MitoXpress Xtra Oxygen Consumption AssayInstrument setup guide for Perkin Elmer plate readersInstrument setupoptimized for each specific plate reader model. The counting delay and window times should not be adjusted from these defined values./chemFor Research Use Only. Not for use in diagnostic procedures.RA.434849537This information is subject to change without notice.© Agilent Technologies, Inc. 2018, 2019, 2020 Printed in the USA, October 1, 2020 5994-0530ENRecommendations for EnVision plate readerThe Perkin Elmer EnVision plate reader with TRF capability allows the use of the MitoXpress Xtra Oxygen Consumption Assay with single-read TRF detection. The TRF intensities represent the level of oxygen in each sample.For optimal performance, we recommend setting up the measurement conditions for Perkin Elmer EnVision reader in the instrument control software according to the following instructions.1. Create a TRF Label in the Wallac Envision Manager.2. Edit the Properties of the Label to set the parameters asdescribed in the following instrument setup table.3. Create a Protocol in the Envision Manager and select touse the new Label.Please refer to the Perkin Elmer instrument software user manual for furtherinformation.Instrument setup* Optimal flash number and measurement height can differ, and should beoptimized for each specific plate reader model. The counting delay and window times should not be adjusted from these defined values.。