From Process Development To Scale-up Facing The Challenges .

Facing the Challenges in Vaccine Upstream Bioprocessing Scalable Production of AAV VectorsHowever, achieving preclinical efficacy testing,especially in large animal models and toxicologystudies, requires vector quantities that simplycannot be produced in a laboratory setting or inmost research-grade vector core facilities. Currentmethods for transfection require use of adherentHEK 293 cell cultures, expanded by preparingmultiple culture plates. Ideally, a single large-scalesuspension culture would be a replacement formultiple culture plates.In this tutorial, we examine some of the currentlyavailable schemes used in generating rAAV fromsuspension cultures, and describe what it takes toachieve scalable rAAV production.Scalable ProductionTwo basic systems for growing cells in cultureexist: monolayers on an artificial substrate(i.e., adherent culture) and free-floating inculture medium (i.e., suspension culture).rAAV vector production uses a triple transfectionmethod performed in adherent HEK 293 cells,which is the most common and reliable method10 GENengnews.com(Lock et al., 2010), albeit resource intensive.Due to its scalability and cost, rAAV cell suspensions are more desirable.To simplify scalability and dramatically decreaseoperational costs and capital investments, useof bioreactors provides process simplification,from pre-culture to final product. Two examplesare the iCELLis from Pall Life Sciences, designedfor adherent cell culture applications, and theWAVE Bioreactor from GE Healthcare LifeSciences, ready for batch culture, fed-batchculture, perfusion culture, and cultivation ofadherent cells.Both are designed for convenient handlingand control of cell cultures up to 25 L. Bothenable rapid and scalable rAAV production.Recently, Grieger et al. (2016) showed suspensionHEK293 cell lines generated greater than 1 105vector genome-containing particles (vg)/cellor greater than 1 1014 vg/L of cell culture whenharvested 48 hours post-transfection, a protocoldeveloped and used to successfully manufactureGMP Phase I clinical AAV vectors.Large-scale productions require consistent andreproducible. AAV produced for clinical usesmust be thoroughly analyzed to identify themain purity, potency, safety, and stability factorsdescribed below.PurityEmpty capsids typically take 50–95% of the totalAAV particles generated in cell culture, dependingon specific serotypes and protocols used. Emptycapsids may solicit deleterious immune responseagainst AAV (Zaiss and Muruve, 2005). It is desirable they be minimized during production andremoved during purification.AAV empty capsids are composed of an AAVcapsid shell identical to that of the desiredproduct, but lacking a nucleic acid moleculepackaged within. Gradient ultracentrifugationusing iodixanol is effective in separating emptycapsids. Assessment and measurement can bedone by either electron microscopy or A260/A280spectrometry. It may be difficult to distinguishAAV capsids containing small fragments of DNA

Facing the Challenges in Vaccine Upstream Bioprocessing Scalable Production of AAV Vectorsnot readily distinguished from completely emptycapsids using density centrifugation or electronmicroscopy.dual-labeled hybridizable probe for detectionand quantification of amplified DNA junctionsequence.Helper virus-dependent replication-competentAAV (rcAAV), also referred to as “wild-type” or“pseudo-wild-type” AAV, is an AAV capsid particlecontaining AAV rep and cap flanked by ITR. Thistype of AAV (rcAAV) is able to replicate in the presence of a helper virus.rcAAV DNA sequence titer is calculated by directcomparison to the fluorescent signal generatedfrom known plasmid dilution bearing the sameDNA sequence. A positive signal indicates an intactleft IRT-rep gene junction has been detected andamplified, representing the maximum possiblercAAV contamination level present in the rAAVvector sample being analyzed. It does not indicate whether the DNA sequence is infectious orcapable of helper-virus assisted replication.Though wild-type AAV is unable to replicateautonomously and requires co-infection withhelper viruses, such as adenovirus, the expression of AAV rep or cap from rcAAV present in anAAV vector increases the risk of immunotoxicity invector-transduced tissues. Replication competentrcAAV is a rare ( 10-8) and yet deleterious event.To assess rcAAV generation, target DNA sequencespans left AAV2 ITR D-Sequence and AAV2 repsequence. An intact left AAV ITR-rep gene junctionis a requisite feature for AAV replication to occurin vivo in the presence of a helper virus. The assayemploys sequence-specific PCR primers and a11 GENengnews.comPotencyHigh potency of AAV vectors is achieved by carefully selecting and isolating full capsids. Physicaland functional titers can be measured to assessthe actual potency of AAV production. Physicaltiter measures the encapsidated AAV vectorgenome, a key mediator and indicator of therapeutic effect. Measurement of vector genomes byquantitative real-time PCR is the closest physicalindicator of rAAV vectors. Functional titer isestablished by measuring transgene proteinexpression in a dose-dependent manner,following transduction into appropriate cell lines.SafetySafety concerns comprise infectious agentsused to generate AAV vectors. Mechanismsto inactivate infectious viruses include heatinactivation of adenoviruses and detergentinactivation of enveloped viruses. A completelist of product release tests can include adventitious virus tests of porcine, canine, and bovineviruses (Table).Payload IncreaseDeveloping viral vector comes with key featuresscientists strive for, including large payloadcapacity. With rAAV, the limited packagingcapacity precludes the design of vectors forthe treatment of diseases associated with largergenes; AAV has a packaging capacity of up to4.5 kb for packaging foreign DNA.

Facing the Challenges in Vaccine Upstream Bioprocessing Scalable Production of AAV VectorsTable. Method of removal and measurement of AAV vector production impuritiesAAV Vector ProductionImpurityMethod of RemovalMethod of MeasurementResidual host cell DNA/RNA(nuclease-sensitive)Nuclease treatment(Benzonase)qPCR using amplicons to generic host cell genome(e.g., 18SRNA gene); qPCR using amplicons forsequences of specific concern (e.g., AdE1); qPCRusing amplicons for non-vector genome sequencesResidual host cell proteinUltracentrifugation; ionexchange chromatographyELISA using polyclonal antibodies detecting representative proteinsResidual plasmid DNA(nuclease-sensitive)Ultracentrifugation;ion exchangechromatographyqPCR using amplicons for helper virus sequences;infectious titer of helper viruses; ELISA or Westernblotting for helper virus proteinsResidual helper viruses(nucleic acids and proteins)Nuclease treatment(Benzonase)Various, depending on componentAAV empty capsidsUltracentrifugation;ion exchangechromatographyElectron microscopy; spectrophotometryEncapsidated hostcell nucleic acids(nuclease-resistant)qPCR using amplicons to generic host cell genomesequencesTable continued on page 1512 GENengnews.comFigure 1. Elements in optimization.Before optimization, the trans-splicingefficiency is about 1–5% when comparedto GFP expression from single vector.After optimization, the efficiency reachesabout 25–50%.

Facing the Challenges in Vaccine Upstream Bioprocessing Scalable Production of AAV VectorsTable(con’t). Method of removal and measurement of AAV vector production impuritiesAAV Vector ProductionImpurityMethod of RemovalMethod of MeasurementqPCR using amplicons to specific sequencesof concern (e.g., E1A)Encapsidated helpercomponent DNA(nuclease-resistant)replication-competent AAVnoninfectious AAV particlesqPCR using amplicons for helper backbonesequencesOther, including aggregated,degraded, and oxidized AAVvectorsAd-dependent amplification; Ad-dependentinfectivity in susceptible cells; various, includingsize-exclusion chromatography, dynamic lightscattering, electrophoresisUtilizing the split vector system, which exploitshead-to-tail concatamerization formation, hasbeen developed to circumvent AAV small packaging capacity. Two main approaches includetrans-splicing and homologous recombinationmethods; both depend upon recombinationbetween two vector genomes, with each genomeencoding approximately half the transgene,within the same cell to achieve gene expression.ViGene has developed a trans-splicing systemthat can expand the payload to 8 kb.13 GENengnews.comWe setup a screen for a more efficient transsplicing system. Here, the GFP reporter was splitand cloned into two AAV vectors. Based on GFPexpression, after co-transfection in HEK-293 cells,we scored expression efficiency of the combination of different trans-splicing elements andincluded the selection of a splicing donor andacceptor sequence, and the annealing sequence inthe intron. Our best vector generated about 70%expression compared to single vector (Figure 1).

Facing the Challenges in Vaccine Upstream Bioprocessing Scalable Production of AAV VectorsWe generated and purified both vectors in AAV9trans-splicing and efficiency and expressionlevels were tested in vitro and in vivo. Following72-hours transduction in HEK-293 cells by AAV9virus (Figure 2), GFP expression was detectablein approximately 70% of cells, about 50% whencompared to single vector GFP expression.Similar efficiency and expression levels wereobserved in RGC neurons, following two-weekintraocular injection.A deeper understanding of the molecular basisfor inherited and acquired diseases continuesto drive the broader adoption of AAV as thevector of choice for treating many diseases.Numerous Phase I and II trials utilizing AAVhave been performed for various inheritedand acquired diseases.A continuation to greatly increase AAV vectoryield, improve AAV potency and purity, andincrease payload size will further make AAVa bigger player in gene therapy. n14 GENengnews.comFigure 2. Trans-splicing AAV vector expression of GFP in vitro and in vivo.Eva Szarek, Ph.D., is scientific marketingand sales managerJeffrey Hung, Ph.D. (jhung@vigenebio.com),is chief commercial officer and GM of cGMPbusiness at ViGene Biosciences.

ROUNDUPIntegrated ContinuousManufacturing of Biologics:Trends in the FieldBiosimilar Manufacturers, Take Note:This Stream Is for YouADDITIONAL CONTENTRandi Hernandez15 GENengnews.comDuring upstream bioprocess developmentbioprocess engineers need to decide between abatch, fed-batch or continuous/perfusion process.Analyzing the benefits of their respective usage atbench scale allows confident process decisions.Watch Webinarreptile8488/Getty ImagesAlthough estimates vary slightly, many industry experts predict continuous manufacturing will, at the least, cut the cost of manufacturing biologics in half. Thus, it will be anattractive option for drug makers looking to trimmanufacturing budgets. But not all biologicswould be feasible candidates for a truly integrated processing stream, and a truly continuous linemay require a larger initial capital investment.To learn more about which companies andproducts will likely incorporate continuousmanufacturing first—and to understand which“hot-button” questions regarding implementation still require attention and clarification—GENspoke to pioneers in the field of continuousbiomanufacturing, including MassimoMorbidelli, Ph.D., professor of chemical reactionengineering at the Institute for Chemical andBioengineering at ETH Zürich; Andrew Zydney,

Facing the Challenges in Vaccine Upstream Bioprocessing Integrated Continuous Manufacturing of Biologics: Trends in the FieldPh.D., distinguished professor of chemical engineering, The Pennsylvania State University;Michelle Najera, Ph.D., downstream development scientist, CMC Biologics; Gerard Gach, chiefmarketing officer, LEWA-Nikkiso America; DanaPentia, Ph.D., senior application scientist, andJohn Bonham-Carter, director of upstream salesand business development at Repligen; GerbenZijlstra, platform marketing manager, continuousbiomanufacturing, Sartorius Stedim Biotech; andKarol Lacki, Ph.D., vice president of technologydevelopment at Avitide.Common conclusions from the experts were thatthe use of surge tanks in continuous lines hasboth benefits and drawbacks (see the Bioprocessing Perspectives column on page 22 in theSeptember 15 issue of GEN); enzyme-replacementproducts and monoclonal antibodies (mAbs) willlikely be the first product candidate types selectedfor integrated continuous manufacture (morespecifically, mAb-based biosimilars); and manufacturers are less likely to switch legacy productsfrom existing batch processes to continuousoperation. The high prices of affinity resins are16 GENengnews.comnot expected to decline significantly in the nearfuture, and continuous production could result inresin cost savings—but the experts explain thereare many benefits of continuous production ofbiologics besides those related to cost.Most of the products that would likely be madein integrated continuous lines will be new products, and will also primarily be labile biologics orthose which have uncertain demand. Continuousoperation, says Bonham-Carter, will allow engineers to react to fluctuations in product demandand give companies the option to build late (orbuild out) significantly, if required. Dr. Morbidelliasserts that peptides, fusion proteins, scaffoldswith mAbs, and other products “containing sensitive antennary glycostructures that are needed forbiological activity” would also be types of therapies that could be made in continuous flows.To tell which pharma manufacturers are likely tointegrate end-to-end continuous manufactureinto production lines first, investors and industryinsiders should follow company patent filingsand peer-review articles authored by companyrepresentatives. As Dr. Zydney points out, manu-facturers that are already “clearly interested” infully continuous biomanufacturing lines includeGenzyme, Merck, Bayer, and Sanofi, amongothers. Also doing work in this space: NovoNordisk, Novartis, Amgen, Shire, Pfizer, WuXi,and BiosanaPharma. In addition, some contractmanufacturing organizations (CMOs) are in thecontinuous biomanufacturing field (such as CMCBiologics), and these organizations are poised tohelp biopharma clients lower the cost to clinic byway of fully continuous manufacturing strategies.Regardless of who is or is not investigating continuous, “What is definitely a right development trend isthat vendors do support the change and offer technologies that enable continuous operations,” says Dr.Lacki of Avitide, a company that makes affinity resins.“At the end of the day, it will be up to the end user todecide how a process needs to be operated, but thechoice will be made on a thorough assessment ofcommercially available technologies.”GEN: What types of biologic medications arethe best/most feasible candidates for integratedcontinuous manufacture?

Facing the Challenges in Vaccine Upstream Bioprocessing Integrated Continuous Manufacturing of Biologics: Trends in the FieldDr. Najera: Any product with an expensivechromatography resin is a suitable candidate forcontinuous chromatography, especially for earlyphase clinical products, where resin cost can becost prohibitive. Continuous capture is also attractive for high-titer processes, since several smallvolumes can be used instead of a single largecolumn. In fact, facility fit limitations are commonfor high-titer processes ( 5 g/L), and continuouschromatography can help manufacturers avoidthe capital costs associated with procurement oflarge stainless-steel columns ( 80 cm diameter)and associated equipment. Finally, the concept ofintegrated continuous manufacture, (i.e., a fullycontinuous process), would be best suited for awell-established late-phase process with a stablemarket demand where cost-savings are realizedthrough basic efficiencies related to batch versuscontinuous processing.Dr. Zydney: This is a difficult question toanswer—it very much depends upon one’sperspective. In some ways, the ‘best’ candidates17 GENengnews.comIntegrated ContinuousManufacturing of BiologicsKarl Rix, Ph.D.,Head of Business Unit Bioprocess, EppendorfGEN: What types of biologic medications are the best/most feasible candidates for integrated continuousmanufacture?Dr. Rix: Besides for the production of less stable proteinswhich in a perfusion process are constantly harvestedfrom the culture, perfusion processes could be especiallyadvantagous to respond to fluctuations in the market.Scale can be varied by modulating the process time or bychanging the number of parallel production lines. This canbe easier than traditional scale-up of a fed-batch process.GEN: The manufacturers of Prezista (Janssen), asmall-molecule drug, got FDA approval to change itsprocesses to a continuous method. To your knowledge,are any biologics manufacturers looking into a similarmanufacturing change? Do you think manufacturers aremore likely to address the end-to-end manufacture of atotally new product, rather than for an existing product?Dr. Rix: Though there have been examples, it is still quiteunlikely that a manufacturer would change an approvedbatch or fed-batch manufacturing process to continousoperation. Usually, the decision for a process modewill be made in product development. In that phase,influencing variables, including product titer, costs formedia, labor, and equipment, and product quality, can besystematically assessed.GEN: To date, why do you think so few biologicmanufacturers have explored the use of an end-to-endcontinuous line?Dr. Rix: In my view, one issue ist that processdevelopment as well as process monitoring strategiesfor continuous processes are less well establishedthan for traditional fed-batch processes. This includesamong others cell line and media development, onlinemonitoring technologies, and scale-down modeldevelopment. This might increase process developmenttime and risks. And in my opinion, regulatoryuncertainties are still an issue.

Facing the Challenges in Vaccine Upstream Bioprocessing Integrated Continuous Manufacturing of Biologics: Trends in the Fieldfor integrated continuous manufacturing areproducts for which there are particular challengesin using batch operations. For example, a highlylabile product that degrades over time wouldbenefit dramatically from the use of a continuousproce

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