BiopharmaceuticalManufacturing in Single-UseBioreactorsCurrent Status and Challenges from a CDMO Perspective

Dr. Tina Lütke-Eversloh and Peter Rogge

Rentschler Biopharma SE, Laupheim

n ABSTRACT

Given today's fast growing biopharmaceutical market, a broadportfolio of Good Manufacturing Practice (GMP)-compliantmanufacturing options is available from traditional stainlesssteel equipment to various disposable devices. This articlefocuses on benefits and drawbacks of implementing single-useequipment for manufacturing biopharmaceuticals to meetboth process efficiency and regulatory guidelines. Dependingon the type of product and the client's requirements, e. g.provision of material for clinical trials, single-use facilitiesallow fast changeover times and a high degree of flexibility. Onthe other hand, Contract Development and ManufacturingOrganizations (CDMOs) face considerable challenges asso-ciated with single-use plastics such as material integrity andsolid (particulates) or chemical (leachables, extractables)contaminations. In addition, an effective supply chainmanagement is required to ensure continuous availability ofconsumables and consistent operational quality.

n ZUSAMMENFASSUNG

Herstellung von Biopharmazeutika in Single-Use-Bio-reaktoren – Aktueller Status und Herausforderungen ausder Sicht eines DienstleistungsunternehmensIm Hinblick auf den schnell wachsenden Markt bietenleistungsfähige biopharmazeutische Dienstleistungsunterneh-men ein breites Spektrum von Good-Manufacturing-Practice(GMP)-konformen Herstellungsprozessen an. Dabei kommenneben klassischen Edelstahlproduktionsanlagen auch diverseSingle-Use-Systeme zum Einsatz. Dieser Beitrag fasst diewichtigsten Vor- und Nachteile der Implementierung vonSingle-Use-Systemen in der biopharmazeutischen Produktionzusammen. Dabei spielen sowohl die Verbesserung derEffizienz als auch die Gewährleistung der behördlichenKonformität eine wichtige Rolle. Zum einen erlauben Single-Use-basierte Prozesse einen schnellen Produktwechsel in derGMP-Produktionsanlage und damit eine extrem hohe Flex-ibilität. Zum anderen gibt es beachtliche Herausforderungen,die z. B. die Materialbeschaffenheit oder partikuläre sowiechemische Verunreinigungen der Kunststoffmaterialien be-treffen. Weitere Herausforderungen sind die kontinuierlicheBereitstellung der erforderlichen Single-Use-Komponentensowie die Qualitätssicherung durch ein effektives Manage-ment der Zulieferkette.

Introduction

The manufacturing of biopharma-ceuticals constitutes a fast growingmarket segment, and many thera-peutic proteins are currently in clini-cal trials and more sophisticatedmolecules for specialized therapiesare developed. These include mono-clonal antibodies (mAb), enzymes,hormones, fusion proteins and otherrecombinant proteins, which areused for the treatment of severediseases, particularly cancer, auto-immune and genetic disorders. Typi-

cally, modern biopharmaceuticalsexhibit improved potency and phar-macokinetic properties, and thus,can be applied in significantly lowerdoses. Since many companies out-source the process development andmaterial supply for clinical studies,the field of Contract Developmentand Manufacturing Organizations(CDMOs) is growing as well [1, 2].

In this article, single-use andmulti-use equipment for mamma-lian cell cultures and related USPtechniques for the manufacturing oftherapeutic proteins are compared.

Upstream Processing (USP)

In general, the mode of operation ofstirred bioreactors for mammaliancell cultures is comparable for bothstainless steel and single-use vessels[3, 4]. Chinese hamster ovary (CHO)cells are favored hosts for recombi-nant production of therapeutic pro-teins due to the glycosylation pat-terns and excellent cultivation tech-niques. Beside fast and easy fed-batch processes yielding high prod-uct titers, CHO suspension culturescan also be subjected to continuous

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bioprocessing which allows relative-ly high volumetric productivities aswell as protein uniformity and con-sistency [5–7]. At the authors’ com-pany, various USP projects em-ployed continuous cell cultivationwith or without cell retention inboth stainless steel and single-usebioreactors. However, most of thecell culture processes are conductedin the straightforward fed-batchmode with monoclonal antibodiesbeing currently the most frequenttype of biopharmaceutical. In thelast 12 months, more than 50 % ofthe Good Manufacturing Practice(GMP) drug substance batches wereoperated in single-use bioreactors atthe authors’ company.

The USP for biopharmaceuticalmanufacturing does not only in-clude the production bioreactor, butalso the seed train from cell thawingto inoculation and the harvest pro-cedure after cultivation. The sub-cultivation steps are typically con-ducted in single-use shake flasks orsingle-use wave bioreactors as seedtrain. Several stirred tank bioreac-tors of different scales are imple-mented in the upscale cultivationline to generate sufficient cellmasses for inoculating the produc-tion bioreactor.

Different harvest procedures canbe used to remove cells and cell de-bris and finally to provide clarifiedliquid for the first downstream pro-cess (DSP) step. An initial centrifu-gation step is included in most pro-cesses, and single-use centrifugationdevices are available for volumes ofup to 6 000 L. Since cell debris andparticles cannot be completely re-moved, subsequent filtration withporous depth filters is necessary toclarify the harvest broth, followedby a final 0.2-µm filtration. Depend-ing on the process and the facility’sequipment, direct filtration is an al-ternative option [8, 9]. The authors’company’s standard for processesconducted in single-use USP unitscomprises two depth and onemembrane filtration steps withoutcell removal by centrifugation, be-

cause suitable depth filters with sig-nificantly improved capacities arecommercially available by now.

Advantages ofSingle-Use Equipment

The main reasons to prefer single-use equipment are the lower risks ofcross-contaminations, lower clean-ing and sterilization efforts, higherflexibility, faster changeover andlower investment costs. Reducedcomplexity, lower maintenance,time-savings and regulatory con-formity clearly increased the portionof single-use equipment in biophar-maceutical manufacturing duringthe past years [10–12]. In addition,more new devices are developed andexisting technologies are contin-uously improved by the vendorssuch as a single-use bioreactor witha nominal volume of 4 900 L [13].

Moreover, installation of single-use equipment is faster and resultsin a smaller footprint due to the lackof CIP and SIP (cleaning/steriliza-tion in place) requirements. Bioreac-

tors can be customized, extendedand are quickly assembled, which isof special interest if multiple prod-ucts are manufactured in the sameGMP suite. The flexible design ofsingle-use devices also allows modu-lar concepts, which can include ei-ther fully disposable equipment orstainless steel/single-use hybrid so-lutions [14]. With respect to opera-tion, utilities and maintenance, fullsingle-use facilities can reduce theoperating costs for an average mAbprocess by > 20 % and labor costsby > 10 % [15].

Disadvantages ofSingle-Use Equipment

Besides the benefits mentionedabove, single-use devices do alsoface some drawbacks; an overview isprovided in Table 1. First, single-usebioreactors are scale-limited due topressure, gas transfer and mixing is-sues. Only recently, the commonlyused maximum volume of 2 000 Lwas extended to 4 900 L with a work-ing volume of up to 4 000 L [13].

n Table 1

Major advantages and drawbacks of single-use equipment forbiomanufacturing.

Item Single-use Stainless steel

Investment costs low high

Technical complexity low high

Maintenance costs low high

Production scale limited not limited

Consumables demand high medium

Material supply high medium

Cleaning efforts none high

Sterilization time none high

Waste production high medium

Energy consumption medium high

Inventory and storage space medium high

Biological contamination risk low medium

Chemical contamination risk medium low

Product changeover quick slow

Overall processing time quick medium

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© ECV • Editio Cantor Verlag, Aulendorf (Germany)

Wissenschaft und Technik

Biotechnologie

However, scales larger than 2 000 Lare not always required for a CDMO,because modern biopharmaceuti-cals often have a significantly im-proved potency and half-life in thepatient’s body. Hence, clearly loweramounts of product are required forthe therapy and many new biopro-cesses enable good titers and yields,minimizing the need for productionscales of > 2 000 L.

The materials have a consider-able impact on employing a single-use-based bioprocess: the demandof consumables is high and suitablestrategies for logistics (e. g. long de-livery times) and storage (e. g. shelflife) must be carefully evaluated.Since storage of consumables is lim-ited, material integrity plays a piv-otal role with respect to availabilityin the case of breaks or leakages orother malfunctions. In general, thecontinuous supply of material ofconsistent quality must be guaran-teed, which constitutes the core ofthe authors’ company supply chainmanagement conducted in close co-operation with the supplier. Never-theless, having a second materialsupply source (if available) is highlyrecommendable for a CDMO, whichcan either be the same supplier withalternative raw material sources or adifferent supplier.

In addition to particulates origi-nating from the plastic material it-self, seals, gaskets or the like, chemi-cal contaminations might cause se-vere problems for the cell cultureand/or the final active pharmaceuti-cal ingredient (API) [16]. The maincontaminants are differentiated ac-cording to the conditions, which ledto their release into the product-containing liquid: whereas leacha-bles migrate under actual processconditions, extractables migrate in-to the test solution under harsh ex-cessive conditions. A multi-stageevaluation concept for leachablesand extractables is therefore per-formed: after an overall risk assess-ment to evaluate relevant materials,a component-specific risk assess-ment is conducted. Considering

Figure 1: Comparison of costs for biopharmaceutical manufacturing using stainlesssteel and single-use equipment. (a) Distribution of COGs, (b) relation of costs andannually produced amounts of drug substance, (c) relation of costs and total numberof batches per year (source: Rentschler Biopharma SE).

Pharm. Ind. 80, Nr. 2, 281–284 (2018)© ECV • Editio Cantor Verlag, Aulendorf (Germany) Lütke-Eversloh und Rogge • Pharmaceutical Manufacturing 283

product safety as most importantparameter, the supplier’s data areanalyzed and compared to the theo-retical worst-case biomanufacturingconditions. If the theoretical ex-tractable concentration might attaina critical dose, remaining safety con-cerns lead to further investigations.For this, standardized tests forsingle-use equipment were develop-ed in the past few years [17].

Economic Considerations

In addition to balancing the above-mentioned pros and cons, economicaspects are taken into account todecide whether a process is operat-ed in a multi- or single-use bioreac-tor. To illustrate this, a typical fed-batch process of mAb manufactur-ing is given to compare the costs ofusing the 3 000 L stainless steel and2 000 L single-use facilities. The cal-culations include the entire pharma-ceutical production process, i. e.both USP and DSP, to manufacture100 kg of drug substance with a pro-ductivity of 2 g L-1 and a purifica-tion yield of 70 %. Capital invest-ment is not considered, which is es-timated to be 30–35 million eurosfor a new stainless steel unit and20–25 million euros for a single-useconstruction. The costs of goods(COGs) are split into direct and indi-rect labor costs, suite utilization,materials (raw materials, water, buf-fers, etc.), consumables (single-usematerials such as bags, tubing, fil-ters), and external costs (e. g. wastemanagement, maintenance). How-ever, the choice of materials andconsumables used for the processmight significantly impact the costs.As shown in fig. 1a, the cost distribu-tion differs only to a minor degreebetween stainless steel and single-use equipment, whereas materialsand labor constitute the main costsfor both types of facilities.

Conducting 24 batches per yearin the 3 000-L stainless steel facility

yields a price of approximately 250euros per gram drug substance,whereas 280 euros g-1 are estimatedfor 36 batches performed in the2 000-L single-use facility. This priceadvantage of stainless steel over sin-gle-use further improves with in-creasing quantities of drug manu-facturing, which is typically the casefor commercial supply of biophar-maceuticals ( fig. 1b). In contrast, theeconomic benefits reverse in favorof single-use bioreactors if the totalnumber of batches per year is sur-veyed, and these price trends furtherbifurcate with lower numbers ofbatches ( fig. 1c).

Conclusions

Since recently, the popularity of sin-gle-use equipment for biomanufactu-ring highly increased, particularly atsmall- and mid-scale CDMOs. Theauthors’ company decided to operateboth single-use and stainless steelfacilities, enabling a high degree offlexibility. However, there is no gold-en rule for deciding which the mostsuitable facility is for all purposes, be-cause it strongly depends on the bio-process and the customer [18, 19].Both technologies have advantagesand disadvantages with respect tocosts, time and safety.

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Correspondence:Dr. Tina Lütke-EverslohSenior Process Manager, USP ProcessDesign and ValidationRentschler Biopharma SEErwin-Rentschler-Str. 2188471 Laupheim (Germany)e-mail: tina.luetke-eversloh@rentschler-biopharma.com

284 Lütke-Eversloh und Rogge • Pharmaceutical ManufacturingPharm. Ind. 80, Nr. 2, 281–284 (2018)

© ECV • Editio Cantor Verlag, Aulendorf (Germany)

Wissenschaft und Technik

Biotechnologie