Compatibility Of A Mobius Single-use Solution For ADC .

Semi-volatile organic analysisDI-GC/MS analysis for semi-volatile organic compounds(SVOC) identified three compounds in capsules exposedto water (diphenyl sulfone, N-methyl-pyrrolidinone(NMP), hexylene glycol) and one compound (4-metoxy3-penten-2-one) in capsules exposed to 20% DMAc.Diphenyl sulfone is a solvent used to manufacture PES,NMP and hexylene glycol are solvents used to make themembrane. No compounds were detected for capsulesexposed to 20% DMSO. Total amounts per capsule foreach identified compound are shown in Figure 2.Opticap XL5 Capsule withMillipore Express SHC MembraneSemi Volatile Extractables via DI-GC/MSExtractables [mg/Capsule]2.502.001.50Bacterial retention test design and evaluation1.000.500.000.5 h 4 h 8 h 24 h 0.5 h 4 h 8 h 24 h 0.5 h 4 h 8 h 24 hWaterDiphenyl SulfoneHexylene Glycol20% DMAc20% DMSON-Methyl-Pyrrolidinone (NMP)4-Metoxy-3-Penten-2-OneFigure 2: Semi-volatile extractables for Opticap XL5 Capsules withMillipore Express SHC membraneNon-volatile organic analysisHPLC-DAD/MS analysis for non-volatile organic compounds identified one compound, diphenyl sulfone, incapsules exposed to 20% DMAc and in capsulesexposed to 20% DMSO. One compound, NMP was alsodetected for capsules exposed to water. Bothcompounds were also identified in the SVOC testing.Total amounts per capsule for each identified compoundare shown in Figure 3.Opticap XL5 Capsule withMillipore Express SHC MembraneNon-Volatile Extractables via HPLC-DAD/MS3.50Extractables [mg/Capsule]3.002.502.001.501.000.500.000.5 h 4 h 8 h 24 h 0.5 h 4 h 8 h 24 h 0.5 h 4 h 8 h 24 hWaterDiphenyl Sulfone20% DMAc20% DMSON-Methyl-Pyrrolidinone (NMP)Figure 3: Non-volatile extractables for Opticap XL5 Capsules withMillipore Express SHC membrane4The identified extractables from Opticap XL5 Capsuleswith Millipore Express SHC membrane are componentsrelated to either the membrane manufacturing processor gamma irradiation. The concentration of thesecompounds in a filtered pool would be determined bydividing the total amount extracted per capsule, asshown in the graphs, by the volume filtered throughthe capsule during processing. As filter throughputincreases, the extractable concentration in the poolwould decrease. In addition, any pre-use flushing of thefilter would further reduce the extractable concentrationin the pool. The results in this study demonstrate that,even with no pre-use flushing, if a relatively low filterthroughput of 150 L/m2 is achieved, all compoundswould be below 0.10 μg/mL concentration. In addition,some level of clearance of leachables can be achievedduring a subsequent ultrafiltration/diafiltration step, inwhich leachables pass through the membrane of thefiltration device while the product is retained[1].Bacterial retention capabilities of Opticap XL150capsule filters with Millipore Express SHC membranewere evaluated after exposure to 20% dimethylsulfoxide (DMSO), 20% dimethyl acetamide (DMAc)and Milli-Q water (control). Recirculation flow loops forthe DMSO and DMAc exposures, unique to eachsolvent, contained two Opticap XL150 Capsule filterswith Millipore Express SHC membrane. Therecirculation flow loop for the water control exposurecontained one Opticap XL150 Capsule filter withMillipore Express SHC membrane. All flow loops weregamma irradiated at 40-60 kGy prior to testing. Testingconsisted of recirculating 600 mL of solvent in eachflow loop for 20 minutes at a flow rate of 1.5 L/minand subsequent incubation in a 40 C oven for 24 hours.Afterwards, the flow loops were emptied and flushedwith Milli-Q water. Capsules were disconnected fromthe loops and submitted for bacterial retention testing.Each device was challenged with Brevundimonasdiminuta (ATCC 19146 ) which was prepared usinga two-step culture method based on the ASTM F838standardized methodology[2] for B. diminuta culture forqualifying sterilizing-grade membrane by retentiontesting. The retention test was performed using aforward pressure of 30 psi at ambient temperature.The challenge feed was prepared to meet the requiredgreater than 107 CFU (colony forming units) per cm2of effective filtration area. In addition, one new device,unexposed to solvent, was also challenged using thesame method.Complete retention of B. diminuta was observed in allOpticap XL150 samples with Millipore Express SHCmembrane. Durapore 0.45 µm filter, used as a positivecontrol, demonstrated 1.7 LRV passage of B. diminuta,which is consistent with historical data. The results aresummarized in Figure 4 and demonstrate that Opticap XL150 capsule filters with Millipore Express SHCmembrane are fully retentive after exposure to 20%DMSO and 20% DMAc.

Bacterial Retention Test(B. diminuta; 107 CFU/cm2)Log Reduction Value [LRV]11.010.0To Transfer PumpSmart TFF FlowPath Assembly75419.08.0Retentate7.06.0TFF SingleUse SensorFlow Cell6 SamplingPortFeed5.034.03.02.0Recovery Line21.00.020% DMSO 20% DMAcexposureexposureDevice 1WaterNo exposure, Durapore exposure,Negative0.45 µmNegativecontrolfilter,controlPositive controlFigure 5: Schematic of the Test Setup used during solvent recirculationin the Mobius FlexReady Solution with Smart Flexware Assemblies forTFFDevice 2Figure 4: Bacterial Retention Testing Results for Opticap XL150Capsules with Millipore Express SHC Membrane with and withoutexposure to Solvents (upward facing arrow indicates complete Retention)Mobius FlexReady Solution with SmartFlexware and sterile connectorsTest designSolvent exposure to Mobius FlexReady Solution withSmart Flexware assemblies for TFFAll single-use components of the Mobius FlexReadySolution with Smart Flexware Assemblies wereevaluated for compatibility with 20% DMSO (DimethylSulfoxide for molecular biology, Sigma-Aldrich, D8418),20% DMAc (N,N–dimethylacetamide, anhydrous 99.8%,Sigma-Aldrich, 271012) and RO water. The wetted flowpath included a feed container, pump head, SmartFlexware assembly, retentate sampling port, as wellas a sensor flow cell. All wetted materials were singleuse and gamma irradiated. Cassette liners and filtrationdevices were excluded from the setup; instead, asegment of Dow Corning Pharma-80 tubing was usedto create a bypass between the feed and retentateports of the Flexware Assembly and the sensor flowcell was connected in this line. A schematic of the setupand a list of components used during recirculation issummarized in Figure 5 and Table 2.5SingleUseRecycleBagItemPart Description1100L Feed Container Assembly2Single Use Feed Pump Assembly3, 5Dow Corning Pharma-80 Tubing4Single Use Sensor Assembly6Sampling Port Assembly7Smart Flexware AssemblyTable 2: List of Components used during solvent recirculation in theMobius FlexReady Solution with Smart Flexware Assemblies for TFFSix liters of solvent was recirculated through thesystem for 24 hours at room temperature, low flow rate(approximately 1.14 L/min) and low pressure ( 0.3bar). Samples for extractables analysis were collectedat pre-determined time points, with fresh solvent addedafter each sampling to maintain a constant recirculatingvolume. A new flow path assembly was installed foreach solvent.Solvent exposure to Lynx CDR, Lynx S2S and ColderAseptiQuik G sterile connectors flow loopsThree types of sterile-to-sterile connectors wereassembled into flow loops and evaluated forcompatibility with 20% DMSO, 20% DMAc and ROwater. One set of flow loops included one Lynx CDRand one Lynx S2S sterile connector, while the secondset of flow loops included two Colder AseptiQuik Gsterile connectors. A unique flow loop was created foreach solvent and was tested separately. A schematicof the setup and a list of components used duringrecirculation is summarized in Figure 6 and Table 3.

Lynx Connectors Flow LoopItemFeedVessel4Lynx CDR and Lynx S2SSterile Connectors123AseptiQuik G Flow LoopFeedVessel41Two AseptiQuik GConnectorsDescription1SS Feed Vessel2Dow Corning Pharma-65 Tubing3Dow Corning Pharma-80 Tubing4Dow Corning Pharma-50 TubingTable 3: List of Components used during Solvent RecirculationFive hundred milliliters of solvent was recirculatedthrough each of the flow loops for 24 hours at roomtemperature, low flow rate ( 1 L/min) and lowpressure ( 0.3 bar). Samples for extractables analysiswere collected as described in the previous section. Afull list of materials of construction that were exposedto solvent in this study are shown in Table 4.2Figure 6: Schematic of Lynx and AseptiQuik G Connectors Setupused during solvent recirculationMaterial of ConstructionComponentPureFlex FilmFeed container, Smart Flexware assemblySiliconeTubing, Sanitary gaskets, O-rings (Lynx S2S, Lynx CDR), Colder MPX, ColderAseptiQuik GPolysulfoneConnectors (Colder MPX, Lynx S2S)PolypropyleneAdaptors, mixer impeller, disposable pump head, couplersPolyolefin ElastomerFeed container portsHDPERetentate diverter, vortex breaker, sample port, low dead volume teeEPDM RubberMixer impeller retainer, disposable pump head, Single Use sensor flow cellSantopreneDisposable pump headPolyphenylsulfoneSingle Use Sensor Flow cellQuartzSingle Use Sensor Flow cellEpoxySingle Use Sensor Flow cell316 Stainless Steel,Electropolished and passivatedLynx CDR male springGlass filled PolysulfoneLynx CDRPolycarbonateColder AseptiQuik G connectorsTable 4: List of Materials of Construction exposed to Solvent.6

Compatibility evaluationPost-solvent exposure visual inspectionAfter 24-hour solvent recirculation and final sampling,solvent was drained, and all assemblies were flushedthoroughly with RO water. All single-use componentsfrom the TFF system as well as the connector loopswere visually inspected for any physical andappearance changes resulting from solvent exposure.ManualBackpressureValvePXV005Valve bypass testingTwo configurations, shown in Figure 7a and Figure 7bwere tested; the first at a flow rate of 18 L/min andfeed pump discharge pressure of 4 bar; the second at aflow rate of 9 L/min and feed pump discharge pressureof 2 bar. The duration of each test was 10 minutes.Sealing integrity of the valves within the Mobius FlexReady solution clamshell as well as all componentsand connections external to the clamshell wereinspected throughout and after the test.PI001PXV004 PCV001PI001PPPI002XV001 XV002XV901Valve ClosedXV102Flow Rate: 9 LPM Pressure set at 2 barFigure 7b: Valve Bypass Testing, Configuration 2Valve cycling testingValve cycling test was performed after valve bypasstesting. The flow rate was set to 18 L/min and pressurewas set to 4 bar in one section of the flowpath, 2 bar inanother section of the flowpath. While these flow andpressure conditions were maintained, two valves wereeach subjected to 103 open/close cycles. Sealingintegrity of the valves within the Mobius FlexReadysolution clamshell as well as all components andconnections external to the clamshell were monitoredthroughout the test. After the valve cycling test wascompleted, the valve bypass testing was repeated todetermine if sealing integrity was still maintained. Thevalve cycling test setup is shown in Figure 8.PCV101PI401PXV005P001Valve ClosedPI001PXV003XV102XV9017XV003XV102Switching Valves(100 cycles)Line CapPressure set at 2 barPressure set at 4 barFlow Rate 18 LPM, Pressure set at 4 barPPI002XV001 XV002Controlled ValveValve OpenedFigure 7a: Valve Bypass Testing, Configuration 1XV003Valve OpenedValve OpenedP001XV001 XV002XV004 PCV001XV005PI002Valve alBackpressureValvePXV004 PCV001P001Post-solvent exposure functionality testingAfter visual inspection, the Lynx CDR, Lynx S2S andAseptiQuik G connector assemblies were incorporatedinto the feed/retentate bypass loop of the Mobius FlexReady Solution with Smart Flexware Assembliesfor TFF and the functionality of all assemblies wasevaluated via Valve Bypass testing and Valve Cyclingtesting using room temperature water. These testswere developed to qualify that manufactured Flexware Assemblies can achieve the maximum process claimsfor flow, pressure, duration, and valve cycles. While thetesting is typically run on new assemblies, performingthe evaluation after solvent exposure is a rigorous wayto confirm whether the solvent has any detrimentalimpact on process capability.PCV101PI401Figure 8: Valve Cycling Test SetupSU Flow CellLynx CDRand Lynx S2SConnectorsAseptiQuik GConnectors

Extractables testingResultsSamples were collected at pre-determined time pointsfor extractables evaluation and the following testmethods were used for analysis.CompatibilityMetal analysisMetals analysis was performed using ICP-OES(Inductively coupled plasma optical emissionspectroscopy) for 38 metal elements. Metals analysiswas performed only on water samples due to assayinterference with DMSO and DMAc. Solvent is notexpected to be more aggressive than water for metalsextraction. Sample detection limit was in the range of0.02 - 0.03 μg/mL.Volatile and semi-volatile organic analysisVolatile and Semi-Volatile Organic compound analysiswere evaluated with HS-GC/MS (headspace gaschromatography-mass spectrometry) and DI-GC/MS(direct inject gas chromatography-mass spectrometry),respectively. All peaks with a response greater than0.1 μg/mL that were detected in the injection sampleat a level 1.5x or higher than in the associated controlsamples were reported as extractables. Injectionsamples were prepared by concentrating test samples20x prior to GC-MS analysis. Therefore, detectionsensitivity as related back to the test samples was0.005 μg/mL.Non-volatile organic analysisNon-volatile organic compounds were evaluated withLC-MS (liquid chromatography-mass spectrometry)using electrospray ionization positive and negativepolarity modes (ESI ). Post-acquisition,chromatograms were assessed for extractable peaksand the collection of associated mass spectra.Reference standards were analyzed in attempt toidentify unknown peaks. Limit of detection for theanalysis was 0.05 μg/mL.Visual inspection of componentsVisual inspection did not reveal any leaks throughoutthe 24-hour recirculation, and no changes in physicalappearance of single-use components were detected asa result of 24-hour contact with solvents. No cracks,crazing, deformation, discoloration, or “stickiness” ofcomponents was detected.Valve bypass testingNo leaks or bypass were observed in any part of theflow path. Flexware assemblies for Mobius sysytemsand all connectors passed the test before and after thevalve cycling test.Valve cycling testingNo leaks or damage to any of the components weredetected throughout the duration of the test.ExtractablesExtractables calculationsSince the Mobius FlexReady Smart Flexware Assemblies and the sterile connector flow loopsincorporate many different components and materialsof construction, the standard approach of basingextractables on exposed surface area was impractical.Instead, an aggregated approach was taken and theresults of extractables are reported in micrograms ofextractables per system [μg/system] and wereobtained by multiplying the assay result value [μg/mL]by the system volume basis [mL] at the timepoint thatthe sample was collected. The method for determiningsystem volume basis for each system is described below.Mobius FlexReady Solution with Smart Flexware Assemblies System:Total extractables for each assay, each solvent, andeach timepoint are calculated based on the 6L that wasrecirculated in the system. No adjustments were madefor sample volumes that were removed and replacedsince they are small (approximately 3%) in comparisonto the total recirculating volume.Lynx CDR and Lynx S2S Connectors System:Total extractables for each assay and each solvent arecalculated based on the 0.5L that was recirculated plusthe volume of additional solvent that was replenishedafter each timepoint sample. Sample replenish volumewas 180 mL for the water extraction, for 4 sampletimepoints. Sample replenish volume was 120 mL forthe DMSO and DMAc extractions, for 3 sampletimepoints. Table 5 shows the volume basis for eachsolvent and sample point.AseptiQuik G Connectors System:Total extractables were calculated following themethods described in the section for Lynx CDR andLynx S2S connectors.8

Mobius FlexReady Solution with Smart Flexware assemblies for TFF and connectors exposed to waterVolume Basis [mL]0.5 HSample2HSample4HSample8HSample24 HSampleWater5006808601040122020% DMSO500n/a62074086020% DMAc500n/a620740860Table 5: System volume basis for extractables reporting of Lynx CDR,Lynx S2S, and Aseptiquick G connectors for each sampling point andsolvent.Metals analysisOnly traces of sodium and calcium were detected in thesubmitted samples. No metals identified are classifiedas elements to be considered in the risk assessment asoutlined in ICH Q3D Guideline for Elemental Impuritiesand USP 232 Elemental Impurities.Non-volatile organic analysisNo compounds were detected in any system or solvent.Volatile organic analysisHS-GC/MS analysis for volatile organic compoundsindicated no extractable peaks in any system or solvent.Extractables after Exposure to WaterSmart Flexware Assemblies for Mobius systems,Method: DI-GC/MS1801601401201008060402000.5 h4h8h24 hExposure Time2,4 Di-tert-butylphenolD5 SiloxaneCumyl AlcoholFigure 9: Extractables for Smart Flexware Assemblies for Mobius systems, exposed to WaterExtractables after Exposure to WaterSmart Flexware assemblies and Connectors forMobius systems, Method: DI-GC/MS180Extractables [µg/system]Semi-volatile organic analysisDI-GC/MS analysis for semi-volatile organic compoundsdid not identify any peaks above 0.05 μg/mL. A fewpeaks were identified at a concentration of 0.05 μg/mL in the Mobius FlexReady Solution with SmartFlexware Assemblies, Lynx CDR, Lynx S2S andAseptiQuik G connectors exposed to water, 20%DMSO and 20% DMAc, as shown below in Figures 9through 14.As seen in Figure 9, 2,4 Di-tert-butylphenol (degradantof an antioxidant), D5 siloxane (extractables fromsilicone) and 2-phenyl-2-propanol (reported as cumylalcohol) were detected in samples collected from theMobius Smart Flexware Assemblies. Figure 10 showsthat several different siloxanes were detected insamples collected from the AseptiQuik G connectorsexposed to water for 24 hours. No compounds weredetected in samples collected from the Lynx connectors. All individual component levels were below0.05 μg/mLExtractables [µg/system]Solvent16014012010080604020024 hTF2S Assemblies24 hTwo Lynx ConnectorsSystem and Exposure TimeCumyl AlcoholD8 SiloxaneD5 SiloxaneD9 Siloxane24 hTwo AseptiQuik GConnectorsD7 SiloxaneD10 SiloxaneFigure 10: Extractables for Smart Flexware Assemblies andConnectors for Mobius systems, exposed to Water9

Mobius FlexReady solution with Smart Flexware Assemblies for TFF and connectors exposed to 20%DMSOMobius FlexReady solution with Smart Flexware Assemblies for TFF and connectors exposed to 20%DMAcFigure 11 shows that only one extractable compound,2,4 di-tert-butylphenol was detected in samplescollected from Mobius Smart Flexware Assembliesafter exposure to 20% DMSO. At the 24-hour timepoint, the compound was below the assay limit ofdetection. D6 Siloxane was detected in samplescollected from the Lynx and AseptiQuik G connectorsa

Test method ASTM D882-12 Standard Test Method for Tensile Properties of Thin Plastic Sheeting was used for the analysis. Strips of film were die-stamped from flat sections and from areas across the seams of the test .