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Changes to bioprocessing methodologies in the biopharmaceutical industry are being driven bythe need for increased speed, a lower cost of goods (COGs) and greater flexibility. Fasterdevelopment times are required to progress biologics and vaccines more rapidly into clinicaldevelopment and then to market to improve worldwide accessibility

Integrated tools forupstream processintensification: part I

Twinned with this is the need to reduce theCOGs of biologics by 90%, says industry bodyBiophorum Operations Group (BPOG), to makethem affordable for growing numbers of peopleglobally.1 These two drivers have led to therequirement of greater flexibility when designingmanufacturing facilities that can accommodate agrowing range of different types of biologics andcan be reconfigured easily to accommodate thehigh attrition rates of monoclonal antibody (mAb)or gene therapies in clinical trials (only one in 10mAbs and even fewer gene therapies will be

successfully licenced for patient use). To meetthese challenges, the biopharmaceutical industryis adopting strategies such as intensified andcontinuous processing that regulatory bodies suchas the US FDA are endorsing as they believe thiswill help to achieve higher product quality whilemaking new therapies more affordable. For intensified and continuous bioprocessing to

deliver on these quality and productivity promises,there is a requirement for intelligently designedproducts; Sartorius Stedim Biotech (SSB) has, inpartnership with the biopharmaceutical industry,

example, an N-3 culture in a RM50 bioreactor(5–25 L working volume) can be used to directlyseed a 500 L bioreactor to skip the N-2, 200 Lbioreactor stage; or, an N-1 500 L culture in astirred or RM bioreactor can be used to seed aproduction culture with a final volume of 2000 Lat a high inoculation density. These different seedtrain intensification approaches are shown inFigure 1.Intensification approaches can also be used in

the production bioreactor. A hybrid approach,whereby the seed train is run in perfusion modewith an N-1 500 L culture being used to seed themain bioreactor (2000 L) with a high cell density,which is then run in fed batch mode to purifyproduct from the bioreactor, is a platformapproach being used by companies includingBristol-Myers Squibb.

developed a toolbox of technologies that arediscussed in detail in this article.

OverviewCurrently, the biopharmaceutical industry isconcentrating on three areas in which upstreambioprocessing can be intensified. The first is in theseed train; here, intensification can be achieved bydirectly inoculating cells from high cell density cellbanks into rocking motion (RM) bioreactors and isa strategy that biopharmaceutical companies areimplementing as it eliminates the need to useshake flasks in the post-thaw expansion phase.Also in the seed train, perfusion approaches —whereby cells stay in the exponential growthphase throughout the entire culture — are beingused to skip steps or generate higher cell densitiesto inoculate the production bioreactor. For

“For intensifiedand continuousbioprocessing todeliver onquality andproductivitypromises, thereis a requirementfor intelligentlydesignedproducts

BIOPROCESSING

July/August 2019 manufacturing chemist 23

Viable cell density of Cellca CHO cells cultured in fed batch and intensified batch conditions (SF = simulated perfusion in shake flasks;golden batch is average fed batch trajectory)

Figure 2

An overview of the different approaches to process intensification

Figure 1

Fully closed seed train

N-2 perfusion

24 manufacturing chemist July/August 2019

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Other companies have installed or aredeveloping platforms to run their productionbioreactor in perfusion mode and either retain theproduct in the bioreactor for batch harvesting(Amgen) or purify their product from thepermeate (Pfizer and WuXi AppTec). However,process intensification can present a problem ifand when cell densities in the bioreactor becomeso high that traditional separation technologiescannot remove all the cells and cellular debris. Asa result, different types of separation technologiesmay be required to separate or harvest the cellsfrom the permeate using a retention system.

Cell lines, media platform and cell bankingFor process intensification in the seed train ormain bioreactor, cell lines that can be cultured inperfusion conditions are required and SSB offersthe Cellca CHO Expression Platform for thisapplication. To demonstrate the performance ofthese cells under perfusion conditions, Cellca CHOcells expressing a model mAb (IgG1) antibodywere cultured in fed batch and simulatedperfusion conditions with a shaker-intensifiedbatch simulation protocol using dailycentrifugation and medium replacement withstandard Sartorius cell culture media and feed mix(1 volume/day perfusion for 15 days). Using the fed-batch culture conditions, 25 x 106

cells were produced, whereas a higher cell densityof 30 x 106 cells was achieved using the simulatedperfusion conditions. This cell density wasdeliberately restricted by a temperature drop-induced cell growth arrest (Figure 2). Althoughthe cell density achieved was not significantly

higher, the titres were (Figure 3). With the fed-batch conditions, 3 g/L was produced, whereas theCellca CHO cells cultured using the simulatedperfusion conditions produced a three-fold greatertitre in 12 days and a four-fold titre increase to12–14 g/L in 15 days.2

SSB also offers a cell bank manufacturingservice, which can save time with intensifiedprocessing as the high cell density vials producedcan be used to directly seed RM bioreactors. Thecell banking service, housed in a 260 m2 cleanroomspace, is a Medicines and Healthcare productsRegulatory Agency (MHRA)-accreditedmanufacturing facility holding an InvestigationalMedicinal Products (MIA, IMP) licence. Thefacility uses media qualified in process simulationtrials and an automatic closed single-use vialfilling system, Fill-It, which can fill 500 vials withup to 5 mL at 50 x106 viable cells/mL and reducesthe risk of contamination. The cell banks arecharacterised in accordance with ICH guidelinesin a good manufacturing practice (GMP)-accredited testing facility and are QP Releasedwithin five months of vial initiation. SSB is alsocurrently working on methods of freezing high celldensities in bags as an additional seed trainintensification option.

REFERENCES1. www.biophorum.com/wp-content/uploads/2017/11/Biomanufacturing-Technology-Roadmap.pdf.2. D. Mueller, et al., “A Versatile Toolbox for Rapid Developmentof Intensified CHO Processes,” poster 554 presented at ESACT2019 (www.esact2019.com/wp-content/uploads/2019/05/ESACT-2019-Poster-Abstracts.pdf).

“Any changes topackagingconfigurationsduring theproduct’sjourney areconsidered to becomplianceevents

Titre produced from Cellca cell line cultured in fed batch and intensified batch conditions (SF = simulated perfusion in shake flasks; FB =fed batch at different stirred tank reactor scales)

Figure 3

FOR MOREINFORMATIONGerben Zijlstra Global Technology ConsultantIntensified and ContinuousBiomanufacturingSartorius Stedim Biotechwww.sartorius-stedim.com

Part II of this article will appearin the September 2019 issue ofManufacturing Chemist