Single-Use Technology in Biopharmaceutical Manufacture

By Regine Eibl and Dieter Eibl

This book gives an overview of commonly-used disposables in the manufacture of biopharmaceuticals, their working principles, characteristics, engineering aspects, economics, and applications. With this information, readers will be able to come to an easier decision for or against disposable alternatives and to choose the appropriate system. The book is divided into two parts – the first is related to basic knowledge about disposable equipment; and the second discusses applications through case studies that illustrate manufacturing, quality assurance, and environmental influence.

• Language: English
• Publisher: Wiley
• Release date: Aug 8, 2011
• ISBN: 9780470922767

Book preview

PART I: Basics

CHAPTER 1

Single-Use Equipment in Biopharmaceutical Manufacture: A Brief Introduction

Dieter Eibl

Thorsten Peuker

Regine Eibl

Chapter Contents

1.1 Background

1.2 Terminology and Features

1.3 Single-Use Systems in Production Processes for Protein Pharmaceuticals: Product Overview and Classification

1.4 Implementation of Disposables

1.5 Summary

1.1 Background

The term biopharmaceutical was first used in the early 1980s [1], when recombinant, commercially manufactured insulin, a therapeutic protein for diabetes patients, was introduced. Various definitions for the word biopharmaceutical are now commonly used [2]. In the United States and Europe, the most frequently used definition is that of a pharmaceutical manufactured by biotechnological methods with organisms, or their functional components, which are of biological origin. Under this definition, all recombinant proteins, monoclonal antibodies, vaccines, blood/plasma-derived products, nonrecombinant culture-derived proteins, and cultured cells, in addition to tissues from human or animal origin and nucleic acids, are considered as biopharmaceuticals [3, 4]. The majority of the above are classified as biologicals (or biologics) by regulatory agencies [5]. Traditional pharmaceutical products, such as chemical substances extracted from plants, secondary metabolites from microbial and plant cell cultures, and synthetic peptides, which may not comply with the above definition, are more often considered as non-biopharmaceuticals. Irrespective of differences in definition, recombinant protein pharmaceuticals are considered an important category of biopharmaceuticals, and their production processes and facilities provide the focus for this book.

The most significant protein pharmaceuticals currently available include hormones (e.g., erythropoietin), enzymes (e.g., human plasminogen activator, asparaginase), vaccines (e.g., influenza, human papillomavirus, cancer), and antibodies (e.g., bevacizumab, trastuzumab). Antibodies, which encompass high-value products with inventory costs ranging from hundreds to thousands of dollars per gram, have attained the highest growth rates and represent by far the most important category [5–7].

In most cases, protein pharmaceuticals are produced with transfected mammalian cell lines. During the last few years, Chinese hamster ovary cell lines have increasingly displaced earlier mammalian cell production systems, such as hybridomas, embryonic feline lung fibroblast cell lines, baby hamster kidney cell lines, and Madin–Darby canine kidney cell lines [8, 9]. It is interesting to note that the publication of Crucell’s work confirming that human retinal cells yield antibody titers in the double-digit range [10, 11] resulted in a few biopharmaceutical companies and contract manufacturing organizations (CMOs) switching to PERC.6 technology [11]. On the other hand, some vaccine manufacturers (e.g., GlaxoSmithKline, Protein Sciences, Novavax) utilize insect cells (Spodoptera frugiperda cells, Trichoplusia ni cells) cultivated in conjunction with the baculovirus expression vector system (see also Chapter 17). Further novel approaches include the plant-cell-derived manufacturing of recombinant proteins, such as plant-cell-expressed recombinant glucocerebrosidase and the hemagglutinin neuraminidase protein of the Newcastle disease virus [12–16] (see also Chapter 21). Although posttranslational modifications in yeasts are possible (e.g., Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris), these are not carried out in an identical manner to that in mammalian cells, and, as a consequence, yeasts offer only limited suitability for recombinant protein manufacture. Furthermore, the use of bacteria such as Escherichia coli for recombinant protein production is also severely restricted [17] (see also Chapter 20).

The most suitable production cell line (which is genetically stable or transient and which can preferably be cultivated as a suspension culture in animal component-free and chemically defined medium) is generally selected with particular reference to the cell density and product expression quantity and quality [18, 19], all of which have a significant impact on the cost of goods (COGs). For example, high product expression levels can reduce capital investment (due to the use of smaller production facilities) and the costs associated with facility qualification and facility operation (fewer production runs resulting in savings on culture medium) with consequent reductions in COGs [10]. Additional cost savings and time line reductions can be achieved through the replacement of stainless steel with single-use equipment, as suggested by various studies [20–23]. Indeed, single-use components (also often referred to as disposables) are increasingly preferred worldwide for the development and production of potentially novel protein pharmaceuticals.

A brief overview of disposables based on their application, a summary of their principal features, and a description of the process flow of a typical production process for a protein pharmaceutical provide an introduction to the subjects of all subsequent chapters. A classification of disposables and the main criteria that must be considered prior to their implementation are also summarized in this chapter.

1.2 Terminology and Features

As the terms disposable and single-use imply, such systems are only ever used once. The disposable systems currently in use have their origins in the fields of medical care (e.g., rubber gloves, sterile swabs, and the technology of intravenous applications) and baby care (e.g., paper towels, disposable diapers). With the exception of special protective clothing and consumables (e.g., swabs, paper towels), single-use products are typically fabricated from plastics approved by the Food and Drug Administration (FDA) (see also Chapters 10 and 11), such as polyethylene (PE), polystyrene (PS), polytetrafluoroethylene (PTFE), polypropylene (PP), or ethyl vinyl acetate (EVA). These materials are typically supplemented with additives to aid performance and/or prolong usable life [24, 25], thereby ensuring their suitability in biopharmaceutical manufacturing applications. In all cases, the product contact surfaces are free of animal-derived components (ADV).

Disposables can be rigid (molded systems) or flexible (bags made from multilayer films) and are often supplied presterilized, having been gamma irradiated at dose levels between 25 and 50 kGy [26, 27], although some are autoclaved. This eliminates the need for subsequent sterilization of the equipment, such as the steam sterilization normally required for stainless steel components. Disposables can therefore be quickly brought into operation. On completion of the process operations, the disposables used are decontaminated and discarded. Thus, time-consuming and expensive cleaning procedures, which may require the use of corrosive chemicals (which could potentially pose a health hazard to the operator) and water for injection (WFI), often considered as a bottleneck in traditional biopharmaceutical facilities, are no longer required.

Disposable technology is often regarded as greener, due to the reduced requirements for cleaning and sterilization. Furthermore, equipment turnaround time is reduced, and process and product changes can be more easily accommodated (a particular advantage in the manufacture of multiple products) when neither cleaning nor sterilization is required [27]. Similarly, the potential for product cross-contamination and microbial contamination are reduced, while the requirements for validation and in-process documentation are minimized [25, 28, 29]. Further benefits of disposables include the saving of time (e.g., development time, manufacturing time, time to market), cost reductions (e.g., capital investment, COGs), and a reduction of the facility’s footprint. It can be concluded that disposables offer distinct advantages compared with their reusable counterparts. They can be smaller, safer, greener, faster, and more flexible while offering savings both in terms of capital outlay and operating costs (see Table 1.1 and Chapter 8).

Table 1.1 Summary of advantages and limitations of single-use equipment

There are of course limitations to the use of disposable systems, due primarily to the chemical, biological, and physical properties exhibited by the polymer material in contact with process fluids and the intermediate or the final product. Nevertheless, the primary risk associated with the use of disposables is the potential migration of undesired components from the plastic material (see also Chapters 10, 11, and 13). The undesired contaminants may either be leachables (which may migrate naturally over time) or extractables (which may migrate when exposed to aggressive process conditions such as high temperatures) [30–34]. Additional issues that limit the use of disposables are restricted scalability (due to the mechanical strength of the material) and the limited availability of single-use sensors (Chapters 6 and 24) and peripheral elements such as valves, connectors, and fittings (Chapter 5) [35–37]. The replacement of the disposable components constitutes an increase in operating costs and contributes to the increased cost of solid waste disposal and consumables (Chapter 14). With the exception of special applications such as high-throughput screening (Chapters 7 and 23), disposables currently preclude the use of advanced automation techniques. Furthermore, it is worth noting that extra training of staff may be necessary as the capacity of a manufacturing facility incorporating disposables increases. A thorough investigation is required to determine whether the benefits of disposable systems are sufficient to overcome their disadvantages in any particular manufacturing scenario (see also Section 1.4 and Chapters 12 and 30).

1.3 Single-Use Systems in Production Processes for Protein Pharmaceuticals: Product Overview and Classification

As illustrated in Figure 1.1, a typical process for the manufacture of a drug product (DP), such as a protein pharmaceutical, includes four main processing stages: (1) the upstream processing, (2) the downstream processing, (3) the final formulation and filling, and (4) the labeling and packaging. In the upstream processing stage, culture media and buffers are prepared (mixed, sterilized by 0.1- and 0.2-µm filters, stored, and transported), seed and inoculum train are produced, and, finally (see Fig. 1.1), a so-called active pharmaceutical ingredient ([API] see also Chapter 13) is expressed in the production bioreactor. The API that is, with only a few exceptions such as membrane proteins, normally secreted into the culture broth has to be separated from cells and clarified after harvesting. The subsequent downstream procedures [38–44], which produce a drug substance (DS), ensure the reduction of product impurities (e.g., protein A, host cell proteins, desoxyribonucleic acid, aggregates) to an acceptably low level and include virus clearing (inactivation and removal by filtration). Consequently, the API must be further concentrated, separated, and purified, requiring chromatography processes (affinity chromatography, anion-exchange chromatography [AEX], cation-exchange chromatography [CEX], hydrophobic interaction chromatography [HIC]) and cross-flow filtration (ultra- [UFs] and diafiltrations [DFs]). Liquid storage and transportation, and buffer preparation also form part of the downstream processing stage. The liquid DS solution is formulated through the addition of stabilizers prior to being sterilized by filtration and/or aseptically filled into sterile containers. The DS may also be stored or transported deep frozen prior to the fill and finish operations. The DS is then labeled and packed to become the commercially available DP.

Figure 1.1 Schematic of a typical manufacturing process for protein pharmaceuticals.

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Disposables have already been adopted for upstream, downstream, and filling processes and their related procedures up to mid-volume scale (see also Fig. 1.2). Chapters 2–6 of this book provide evidence that upstream processes can already be completely designed with disposables. In addition to bioprocess containers (BPCs) for buffers, media, and bulk products, single-use mixers, sampling and coupling systems, and disposable bioreactors, which can be wave-mixed, stirred, or, more recently, orbitally shaken and pneumatically driven, are also available.

Figure 1.2 Process flow diagram of a middle-scale production for a protein pharmaceutical in which disposables are used.

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In fact, the lack of fully disposable facilities for manufacturing of protein pharmaceuticals at large scale can be ascribed to the downstream processing (Chapter 8), in particular, the affinity purification step, where no single-use solution exceeding 20 L in size is available [7, 45, 46]. Furthermore, single-use solutions for filling operations have yet to become well established (Chapter 9).

Figure 1.3 provides an overview of the primary disposable products currently utilized in pharmaceutical protein manufacturing [47]. The products can be classified in three groups: expendable laboratory items, simple peripheral elements (stand-alone components), and multi­component systems for unit operations and platform technologies (see Chapters 12, 26, 27, and 28). Whereas expendable laboratory items are devices used in routine laboratory work, flexible tubing, fittings, valves, tri-clamps, and so on, are typical of those disposable products categorized as peripheral elements. Further important peripheral elements, which are also disposable by design, include connectors and couplers, dispensing and sampling bag manifold systems, and simple containment bags for liquid and powder.

Figure 1.3 Primary categories of disposable products used for the development and manufacture of novel biopharmaceuticals

(adapted with kind permission of John Wiley & Sons)

[46].

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Among multicomponent systems used for unit operations, BPCs, disposable membrane adsorbers (see Chapter 25) used for removing high-molecular-weight contaminants by AEX, for example, depth filter systems, and both membrane and wave-mixed bioreactors enjoy the longest tradition of use. Disposable pumps, freeze–thaw systems, centrifuges, and UF/DF systems [47] are relatively recent additions to the disposable product portfolio.

Monge [48] proposed a categorization for disposables that distinguishes between generic, standard, and unique single-use systems (Fig. 1.4) and is based on the specificity of design from a particular supplier.

Figure 1.4 Proposed categorization of disposables according to Monge [48].

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1.4 Implementation of Disposables

Disposables may be used in the same manner as their stainless steel counterparts, provided due consideration is given to their specific characteristics. The user’s requirements constitute the primary criteria in the decision-making process, while the projected product demand and the optimized usage of the asset must also be taken into account. The performance of the disposable, the associated costs, and the security of the supply chain must also be considered, while the risk of using a disposable must be minimized. Disposables are in their infancy, both in terms of their development and their use in commercial manufacturing systems, and thus pose particular challenges in terms of assessing the technical risk associated with their use and the security of their supply chain. It can be concluded that disposables are not well established in all areas of biopharmaceutical production, and, consequently, there is a lack of long-term studies, references, and the corresponding rules and standards that would normally be associated with a mature technology. Furthermore, numerous suppliers offer a broad range of products and technologies with each being at a different development stage. Currently, no supplier is able to provide all available technologies. The majority of products have not been standardized, and, therefore, the security of supply outlined in Figure 1.5 is of paramount importance when considering the utilization of disposables.

Figure 1.5 Questions concerning security of supply.

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The essential prerequisite for the successful introduction and application of disposables in biopharmaceutical manufacturing is a thorough understanding, and the appropriate management, of the associated risks. In this context, numerous factors must be considered. Pora and Rawlings [24] defined 18 critical factors that may be used as a checklist: (1) capital investment, (2) disposable consumable costs, (3) extractables and leachables, (4) area footprint, (5) sterilization methods, (6) utilities supply, (7) chemical compatibility, (8) physical compatibility, (9) solid waste disposal, (10) liquid waste disposal, (11) energy use, (12) labor requirements, (13) scale-up, (14) process flexibility, (15) unit operations, (16) available technology, (17) development lead times, and (18) assurance of sterility.

By way of contrast, Sinclair and Monge [48–51] recommended an integrated approach encompassing the engineering, procurement, and operations groups as a means of evaluating risk, requiring close collaboration with the supplier during the evaluation and implementation processes. The focus of this approach is the maturity of the technology, economics, supplier dependencies, the supply chain, and validation. The task of the operations group is to define user requirements, to identify operational risks, to consider standard versus custom designed systems, to identify potential compromises, and to assess the operability issues associated with the use of disposables. In other words, available materials and their acceptance for biopharmaceutical manufacturing have to be compared, vendor history has to be checked, and the vendor’s documentation on extractable and leachable studies must be reviewed by this group. Finally, the operations group must confirm the need for further extractable and leachable studies and must define the overall cost and develop an implementation plan. The engineering group has the responsibility of identifying the best technology available for the purpose, to design the process for the whole life cycle and to ensure that the designs support the procurement goals, while ensuring compliance with the cost, time, and quality objectives. The procurement group (which should be involved at an early stage) is responsible for developing a secure supplier strategy (in which supplier dependencies are minimized and a second supplier is identified) and integrating capital and operational considerations.

Risk identification and mitigation strategies most often take the form of formal risk and cost assessments, which typically comprise performance testing, extractable and leachable studies, toxicologist and health-based risk assessments, evaluation of life cycle costs for both capital and operations, cost models, and development of standard specifications and designs, coupled with an evaluation and ranking of the suppliers [52–61].

1.5 Summary

This chapter introduces the basic terms associated with disposables and illustrates their importance and their diversity of use in biomanufacturing applications. Whereas disposables were initially used only in research and development (R & D), they are now increasingly used in commercial manufacturing operations and, in particular, during the last 5 years by CMOs. Hybrid production facilities (Chapter 12), incorporating disposable devices from BPCs for buffer, media, and bulk products through mixing systems and disposable bioreactors to single-use equipment for filtration, chromatography, and filling, represent the state of the art today. The development of hybrid facilities, which is expected to continue, is being driven by international healthcare needs on one hand, and achievements in cell engineering, resulting in as much as a 10-fold increase in product titers, on the other.

The ongoing research aimed at eliminating the current limitations of disposables is expected to support this trend. Computational fluid dynamics (CFD) studies (Chapter 22) and verifications, the development of novel single-use sensors (Chapters 6 and 24), the integration of gamma-sterilizable radio frequency identification (RFID) tags [62], and their combination with single-use sensors will all facilitate process traceability and the development and commercialization of fully disposable production facilities. The reader is referred to Chapter 15 for a detailed discussion of the trends arising from the increased usage of disposables.

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CHAPTER 2

Single-Use Bag Systems for Storage, Transportation, Freezing, and Thawing

Nathalie Riesen

Regine Eibl

Chapter Contents

2.1 Introduction

2.2 Bags for Fluid and Powder Handling

2.2.1 Tank liners

2.2.2 Two-dimensional bags

2.2.3 Three-dimensional bags

2.3 Bag Handling and Container Systems

2.3.1 Bag handling systems

2.3.2 Container systems for in-house applications

2.3.3 Container systems for liquid shipping

2.4 Single-Use Bag Systems for Freezing and Thawing

2.5 Summary

2.1 Introduction

Media, starting materials, and intermediate and finished products may have to be sampled, stored, frozen, thawed, transported in-house, or shipped during the manufacture of biopharmaceuticals. Single-use bag systems have become accepted alternatives to the sampling, storage, and transportation systems fabricated from glass or stainless steel, which have been traditionally used in these processes. Their usage is not new and is based on the technology associated with sterile intravenous medical applications, which commonly employ single-use bags, consisting of one-layer films made from polyvinyl chloride (PVC) or ethylene vinyl acetate (EVA), for the storage of blood or infusion solutions. Various bioprocess bags have been used in the research, development, and manufacture of biopharmaceuticals for over a decade [1–3]. Figure 2.1 summarizes the typical fields of application of single-use bag systems in biopharmaceutical production processes. A thorough assessment of the potential risks to the process, product, and/or patient must always be completed prior to the implementation of single-use bags in these numerous applications.

Figure 2.1 Summary of single-use bag applications in biopharmaceutical manufacturing.

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Single-use bags are made from plastic films [4] (Chapter 10), the composition of which strongly influences their construction, performance characteristics, and production capabilities. Modern bioprocess bags are fabricated from multilayer films. The composition and quality of the bag material vary with the bag manufacturer. The customer can select the bag depending on the design, volume, available ports, tubing, in-line filters, and package. The majority of bag manufacturers offer both standard and customer-specific bag solutions.

In this chapter, the most important types of single-use bag systems designed for the handling of fluids and powders are introduced, with particular focus on the characteristics and typical applications of tank liners and two-dimensional (2D) and three-dimensional (3D) bags. In addition, the bag handling and container systems that are, with few exceptions, required for trouble-free use of single-use bags are discussed. Finally, the primary considerations associated with bag-based freezing and thawing of cells, and intermediate and finished products are briefly described.

2.2 Bags for Fluid and Powder Handling

2.2.1 Tank Liners

Tank liners are simple, single-use bags used to line container and transportation systems. In most cases, they are not gamma sterilized. As shown in Figure 2.2, tank liners are used in open, single-use systems in which the bag is placed in a container to form a lining [5]. The sole function of the container is to provide physical support.

Figure 2.2 Thermo Scientific TC Tech Tank Liner in a drum

(with kind permission of Thermo Fisher Scientific).

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The function of the tank liner is to contain the liner content and to create a barrier between the liner content and the container wall and/or environment. Tank liners are well suited for mixing tasks, such as media and buffer preparation or in the formulation of drug products, in which large quantities of solids must be added and dissolved. Commercially available overhead mixers can readily be integrated because these systems are open.

2.2.2 Two-Dimensional Bags

Bags for Fluid Handling

Two-dimensional bags are used when small volumes of liquid must be handled. Liquid volumes between 50 mL and 50 L are typical. Larger bags with volumes up to 200 L are available but are exceptions and are utilized for special applications as in case of the Cellbag 200 L and the CultiBag 200 L (see Chapter 4). These bags are produced from two-layer films, which are welded together at their ends. The result is a flat chamber, which has ports either face welded (Fig. 2.3a) or end welded (Fig. 2.3b).

Figure 2.3 Examples of 2D bags: (a) face-ported bag and (b) end-ported bag

(with kind permission of Meissner Filtration Products).

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The number and positioning of ports and assemblies (tubing, pinch clamps, connectors, filters, etc.) are manufacturer specific and depend on the bag type. Most bag manufacturers offer a number of variations within one standardized series of bag types. As mentioned in the introduction, customized solutions are available. However, they tend to be very expensive because of the additional complexity and cost of manufacturing. The use of customized bags is therefore only likely to be cost-effective if the bags can be used in large quantities.

Two-dimensional bags are utilized either in a reclining or hanging position. In addition to individual single-use bags, multiple 2D bags, for example, for use in manifolds for sampling, dispensing, and product hold, are also available (see Chapter 5).

Bags for Powder Handling

Bags designed for the handling of solids are funnel shaped, flexible, and transparent. They are equipped with large sanitary fittings or aseptic transfer systems (see Chapter 5) at the funnel outlet (see Fig. 2.4), and are antistatic and free of additives. Applications of these powder bags (as they are also known) include closed transfer and storage, as well as transport of dry powder media, buffer salts, pharmaceutically active substances, and adjuvants. They are currently available from various manufacturers in different designs with capacities up to 100 L [5–8].

Figure 2.4 Thermo Scientific HyClone Powdertainer II with clamp and wash down line

(with kind permission of Thermo Fisher Scientific).

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2.2.3 Three-Dimensional Bags

Three-dimensional bags are available for more complex applications and larger liquid volumes in which (as in the case of tank liners) the outer bag container has a support function. Depending on the customers’ requirements, these bags can accommodate liquid up to 2500 L in volume. The 3D design results from the welding of appropriate multilayer films as described in detail in Chapter 10 of this book. The most common designs are cylindrical–conical shaped (Fig. 2.5b) or cube shaped (Fig. 2.5a), versions of which are also available with and without an outlet cone.

Figure 2.5 Examples of 3D bags: (a) TepoFlex biocontainer

(with kind permission of Meissner Filtration Products)

and (b) CultiBag STR

(with kind permission of Sartorius Stedim).

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In contrast to 2D bags, 3D bags offer more flexibility with regard to the positioning of ports. Top, bottom, and/or face-ported 3D bags are available commercially. Furthermore, there is a wide selection of port size and port complexity. Finally, it is worth mentioning that 3D bags form the basis of all larger single-use storage and transportation systems, nearly all single-use mixing systems (except small-volume, wave-mixed systems, see Chapter 3), and all stirred single-use bag bioreactors (see Fig. 2.5b and Chapter 4).

2.3 Bag Handling and Container Systems

2.3.1 Bag Handling Systems

Due to the inherent characteristics of film materials and the welding and adhesive technologies utilized during their manufacture, single-use bag systems have a limited mechanical load capacity. The tensile strength of the films, coupled with the strength of the welded seams, largely determine the maximum mechanical load capacity, and thus the size, shape, and application of the bag. In addition, the material characteristics are strongly temperature dependent. For example, some plastic films may fail in a brittle manner at low temperature.

Apart from small 2D bags used at ambient temperatures, all systems require an outer container. However, even small-volume 2D bags can be difficult to handle, and therefore, a suitable handling system is also recommended for these bags to ensure safe storage and/or transportation. In many cases, simple, stackable trays, or racks made from plastics or stainless steel are suitable for 2D bags; however, very complex systems can also be obtained for specific applications. Simple systems such as stackable trays with carrying handles and inserts for tube guides are shown in Figure 2.6a,b [9, 10]. Optimum accessibility and flexibility are ensured through the use of modular, expandable rack systems (see Fig. 2.6c).

Figure 2.6 Selected bag handling systems for liquids: (a) Plastic Flexboy Trays, (b) Stainless Flexboy Trays, and (c) Flexboy Rack

(with kind permission of Sartorius Stedim).

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Special protection must be provided for bags in which liquids are frozen or frozen liquids are stored and transported. The port/bag joints are of particular concern, but there is also a risk of brittle failure at the tube/fitting transition points. Various manufacturers have therefore developed specific bags, made from special films and correspondingly secure handling systems, which can be used at both low and high temperatures (see Fig. 2.7). Some bag handling systems can even withstand freezing and autoclave temperatures [11–13]. The most suitable single-use bag systems for freezing and thawing currently available are discussed in Section 2.4.

Figure 2.7 Selected bag handling systems for frozen liquids: (a) Thermo Scientific Nalgene Bioprocess Bag Management System

(with kind permission of Thermo Fisher Scientific),

(b) single-use bag protection systems BioShell

(with kind permission of UFP Technologies),

and (c) single-use container for flexible freeze–thaw processes Celsius FFT

(with kind permission of Sartorius Stedim).

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2.3.2 Container Systems for In-House Applications

Bioprocess containers for in-house applications are used if larger and more complex bags must be handled at the place of manufacture (Fig. 2.8a). They are made from stainless steel or plastic and are available for cylindrical–conical-shaped bags and cube-shaped bags but are not intended for shipping. Modern cube-shaped bioprocess containers are stackable and occupy the minimum space necessary (Fig. 2.8b). They can be transported with pallet lifting trucks, forklifts, or dollies. In most cases, containers that exceed volumes of 500 L are operated in situ [9].

Figure 2.8 Bioprocess container for in-house applications: (a) Flexel 3D Palletank for storage and (b) Flexel 3D Palletank for in-process fluid handling

(with kind permission of Sartorius Stedim).

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Bioprocess containers can be equipped with weight sensors, recirculation/mixing fluid management, and temperature control as required. The temperature of the bag content can be controlled through the heating or cooling of the heat transfer fluid circulated within a double jacket.

2.3.3 Container Systems for Liquid Shipping

There is a demand for safe, stable, and closed container systems when sterile liquids are shipped in single-use bags. The factors affecting the selection of suitable systems include the distance of travel, the means of transpor­tation, the temperature sensitivity, the volume, and the possibility of foam formation during formulation [14]. Such systems are referred to as liquid shipping containers. Container systems for liquid shipping can of course also be used for in-house applications, insofar as they meet the demands of the application. It is important to note that some commercially available systems are only suited for usage in noncritical areas.

In particular, in the case of larger volumes, the liquid must be held in position and not allowed to oscillate. Vendors of container systems for liquid shipping, such as Thermo Fisher Scientific and Sartorius Stedim, offer appropriate devices, known as the Smartainer II Shipper and the Flexel 3D, respectively. These devices ensure safe and secure shipping of sterile liquids up to 500 L in volume, as an adjustable cover plate holds the liquid in place and thus suppresses any liquid wave action during the shipping process (see Fig. 2.9).

Figure 2.9 Flexel 3D Palletank for shipping

(with kind permission of Sartorius Stedim).

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2.4 Single-Use Bag Systems for Freezing and Thawing

Frozen, biopharmaceutical intermediate and finished products remain stable over the long term, thereby facilitating storage and transportation. Moreover, the potential for undesired reactions and contamination are minimized, and transportation under controlled and low temperatures is therefore possible [15]. The freezing systems used in biopharmaceutical manufacturing depend on the nature and quantity of the frozen cargo. While small plastic tubes and bags find application at milliliter scale, bags as well as carboys are used at liter scale (10–20 L), and large, transportable, and controlled freezing containers made from stainless steel are utilized in large-scale systems (50–500 L) [13, 16–20].

Single-use bags have proven to be appropriate for the freezing of biologics. The composition of the bags, enabling safe usage at low temperatures, as well as the availability of suitable bag handling systems (see Section 2.3.1) and controllable freezing and thawing apparatus, facilitates high freezing and thawing rates without compromising operating safety [20]. A further benefit of bag systems is the incorporation of manifold filling systems (see Chapter 5). As a consequence, a simple portioning of the liquid can be achieved prior to freezing, thereby facilitating further processing. This permits the subsequent thawing of only the required quantity of product at any particular time. Furthermore, new application fields for bags are being developed, in combination with freezing processes, enabling long-term storage. For example, the procedure for the production of the working cell bank (WCB)-derived seed inoculum can be simplified and shortened. By doing so, larger cell quantities can be filled, frozen, and stored in bags [21]. Moreover, it is conceivable that in the future, bioreactors could be seeded directly with thawed cells previously cryopreserved in a bag. Hence, intermediate cultivation steps and thus time and money could be saved. A potential disadvantage of bags that should not be underestimated, in addition to the recognized problem of leachables and extractables (see Chapter 1), is possible, undetected damage to the frozen bag. Bag integrity cannot typically be confirmed until the contents of the bag have been thawed [22].

Bags suitable for cryopreservation can be frozen and thawed in traditional freezing and thawing systems, but it is not possible to control the freezing and thawing processes if simple devices are used. Controlled freezing and thawing requires the use of advanced freezing and thawing systems, such as the Celsius-Paks System from Sartorius Stedim. This is the first controllable freezing and thawing apparatus for use with bags with working volumes between 30 mL and 16.6 L. When used in combination with a control unit, freeze–thaw modules, transfer carts, storage modules, and shipping units, biopharmaceutical liquids can be filled into bags, frozen under controlled conditions, stored and/or shipped, control thawed, and further processed (see Fig. 2.10) [13, 23].

Figure 2.10 Sartorius Stedim Freeze–Thaw System: (a) 1- and 2-L Celsius-Pak, Celsius-Pak Carrier and 8.3- and 16.6-L Celsius-Pak, (b) FT 100 Freeze-Thaw Module with CU 5000 Thermal Control Unit and Transfer Carts, (c) Celsius Shippable Storage Module (SSM) and SSM Trolley, and (d) Celsius Shipper and Celsius SSM Shipper

(with kind permission of Sartorius Stedim).

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2.5 Summary

The multitude of single-use bag systems for liquid and powder handling available on the market is described in this chapter. The use of 2D and 3D bioprocess bags has become well established in most areas of biopharmaceutical manufacturing including research, development, and production. They have been developed from simple storage devices, typically used up to now, to instrumented bioprocess units [24]. The importance of single-use bags as alternative solutions in traditional sampling, storage, transport, freezing and thawing systems, and bioprocess units is increasing with the growing acceptance of single-use devices and, in particular, the adoption of platform technologies (see also Chapters 12, 27, 28, and 29).

Bioprocess bags provide greater flexibility and enable simpler execution of multiproduct facilities in the biopharmaceutical industry compared with more traditional systems [25, 26]. It is predicted that their acceptance will continue to grow in the future, driven largely by the availability of improved materials and manufacturing technologies and better understanding of the professional handling of single-use bags and their accessories. The standardization of bags and containers is, however, highly desirable, in particular to facilitate vendor replacement, but progress toward this goal has so far been limited.

References

[1] Haughney H, Hutchinson J. (2004). A disposable option for bovine serum filtration and packaging. BioProcess Int. Suppl. 4(9):2–5.

[2] Wong R. (2004). Disposable assemblies in biopharmaceutical production: Design, implementation and troubleshooting. BioProcess Int. Suppl. 4(9):36–38.

[3] Eibl R, Eibl D. (2009). Application of disposable bag bioreactors in tissue engineering and for the production of therapeutic proteins. In C Kasper, M van Griensven, R Pörtner (eds.), Bioreactor Systems for Tissue Engineering, Series: Advances in Biochemical Engineering/Biotechnology, Vol. 112. Berlin; Heidelberg: Springer, pp. 183–207.

[4] Barbaroux M, Sette A. (2006). Properties of materials used in single-use flexible containers: Requirements and analysis. Avail­able: http://biopharminternational.findpharma.com/biopharm/article/articleDetail.jsp?id=423541&sk=&date=&pageID=7. Accessed December 28, 2009.

[5] Scientific HyClone BPC® Products and Capabilities. (2008/2009). Catalog.

[6] ATMI Products. (2009). Catalog.

[7] Sartorius Stedim. (2009). Products process: Bags, containers & fluid management systems. Available: http://www.sartorius-stedim.com/index.php?id=6495. Accessed December 8, 2009.

[8] DoverPac® SF. (2009). Available: http://www.doverpac.com/products_doverpac_sf.cfm. Accessed December 28, 2009.

[9] Bean B, Matthews T, Daniel N, Ward S, Wolk B. (2008). Guided wave radar at Genentech: A novel technique for non-invasive volume measurement in disposable bioprocess bags: GWR may be a cheaper, more practical alternative to traditional methods. Available: http://www.pharmamanufacturing.com/articles/2008/185.html. Accessed December 8, 2009.

[10] Wang E. (2006). Cryopreservation, storage and transportation of biological process intermediates. BioProcess Int. Industry Yearbook 2006:78–79.

[11] Thermo Fisher Scientific. (2009). Thermo Scientific Nalgene Bioprocess Bag Management System. Available: http://www.nalgenelabware.com/features/featureDetail.asp?featureID=70. Accessed December 28, 2009.

[12] UFP Technologies. (2009). BioShell™. Available: http://www.bio-shell.com/gallery.html. Accessed December 28, 2009.

[13] Sartorius Stedim. (2009). Products process: Freeze and thaw systems. Available: http://www.sartorius-stedim.com/index.php?id=9547. Accessed December 28, 2009.

[14] Lok M, Blumenblat S. (2007). Critical design aspects of single-use systems: Some points to consider for successful implementation. BioProcess Int. Suppl. 5(5):28-31.

[15] Singh SK. (2007). Storage considerations as part of the formulation development program for biologics. Am. Pharm. Rev. 10(3):26–33.

[16] Goldstein A, Loesch J, Mazzarella K, Matthews T, Luchsinger G, Javier DS. (2009). Freeze bulk bags: A case study in disposables implementation: Genentech’s evaluation of single-use technologies for bulk freeze-thaw, storage, and transportation. Available: http://biopharminternational.findpharma.com/biopharm/Disposables+Articles/Freeze-Bulk-Bags-A-Case-Study-in-Disposables-Imple/ArticleStandard/Article/detail/637583. Accessed December 28, 2009.

[17] Zeta. (2009). FreezeContainer®. Available: http://www.zeta.com/DE/Produkte/Freeze-Thaw-Systeme/. Accessed December 28, 2009.

[18] Singh SK, Kolhe P, Wang W, Nema S. (2009). Large-scale freezing of biologics: A practitioner’s review, part 1: Fundamental aspects. BioProcess Int. 7(9):32–44.

[19] Singh SK, Kolhe P, Wang W, Nema S. (2009). Large-scale freezing of biologics: A practitioner’s review, part 2: Practical advice. BioProcess Int. 7(10):34–42.

[20] Rathore N, Rajan RS. (2008). Current perspectives on stability of protein drug products during formulation, fill and finish operations. Biotechnol. Prog. 24(3):504–514.

[21] Scott C. (2007). Single-use bioreactors: A brief review of current technology. BioProcess Int. Suppl. 5(5):44–51.

[22] Lam P, Sane S. (2007). Design and testing of a prototype large-scale bag freeze-thaw system: The development of a large-scale bag freeze-thaw system will have many benefits for the bio­pharmaceutical industry. Available: http://biopharminternational.findpharma.com/biopharm/Disposables/Design-and-Testing-of-a-Prototype-Large-Scale-Bag-/ArticleStandard/Article/detail/473322. Accessed December 28, 2009.

[23] Weidner J, Jimenez F. (2008). Scale-up case study for long term storage of a process intermediate in bags. Am. Pharm. Rev. Available: http://www.americanpharmaceuticalreview.com/ViewArticle.aspx?ContentID=3486. Accessed January 13, 2010.

[24] DePalma A. (2006). Bright sky for single-use bioprocess products. Genet. Eng. Biotechnol. News 26(3):50–57.

[25] Langer ES, Price BJ. (2007). Biopharmaceutical disposables as a disruptive future technology. BioPharm Int. 20(6):48–56.

[26] Williamson C, Fitzgerald R, Shukla AA. (2009). Strategies for implementing a BPC in commercial biologics manufacturing. BioProcess Int. 7(10):24–33.

CHAPTER 3

Bag Mixing Systems for Single-Use

Sören Werner

Matthias Kraume

Dieter Eibl

Chapter Contents

3.1 Introduction

3.2 The Mixing Process: Its Definition and Description

3.2.1 Mixing quality

3.2.2 Mixing time

3.2.3 Residence time distribution

3.2.4 Reynolds number

3.2.5 Specific power input

3.3 Single-Use Bag Mixing Systems

3.3.1 Classification

3.3.2 Bag mixing systems with rotating stirrer

3.3.3 Bag mixing systems with tumbling stirrer

3.3.4 Bag mixing systems with oscillating devices

3.3.5 Hydraulically driven bag mixing systems

3.4 Summary and Conclusions

3.1 Introduction

Mixing constitutes one of the most critical process operations used in the manufacture of biopharmaceuticals [1] and is utilized to achieve homogenization, suspension, dispersion (liquid–liquid and gas–liquid), and heat exchange. Such unit operations are often necessary in medium and buffer preparation, cell cultivation procedures, during fermentations, in final formulation, and in the filling of biologicals. Mixing procedures may also be required to avoid sedimentation and demixing, or to achieve temperature shifts during harvest, concentration, purification, formulation, and filling [2–4].

Conventional mixing devices fabricated from stainless steel currently represent the gold standard in biopharmaceutical manufacturing. However, the increasing interest in disposable technology within the biopharmaceutical industry, due to its numerous advantages [5–13] and the fewer constraints [14] compared with conventional steel, has resulted in the development of single-use bag mixing systems. The customer can currently choose between numerous, single-use bag mixers, which differ in their scale, mixing principle, and their level of inherent cleanliness, instrumentation, and automation.

Following a brief introduction to the main engineering aspects of mixing processes, the application of mixing in the manufacture of biopharmaceuticals is discussed. Single-use bag mixing systems are categorized, and the design features, working principles, advantages, limitations, and potential applications of commercially available, single-use bag mixers are described and discussed.

3.2 The Mixing Process: Its Definition and Description

The process of mixing constitutes the distribution of solid or fluid elements in a volume in which these elements differ in at least one property. The properties are defined by concentration, aggregation, particle size and shape, drop size and shape, temperature, viscosity, color, density, and so on. The aims of mixing are the realization of a specific mixing degree and the production of an intermediate or end product. Moreover, mixing is a prerequisite for some subsequent reactions and can facilitate both heat and mass transfer [15, 16]. Mixing can be divided in the categories distributive mixing, dispersive mixing, and diffusive mixing. Distributive mixing is the adjustment of properties leading to spatial uniformity of all components. Dispersive mixing is defined as the disintegration of agglomerates or lumps to the desired ultimate grain size of the solid particulates or the domain size (drops) of the immiscible fluids [17]. In the latter, the existing stable agglomerates are broken up. In contrast, diffusive mixing (which is less relevant in industrial processes) is characterized by an equilibrium concentration resulting from molecular diffusion [15]. Laminar mixing, often encountered in fluids with high viscosities, originates from a longitudinal mixing where fluid motion is dominated by linear viscous forces. Fluid particles flow along parallel streamlines in a time-independent manner. To achieve homogeneity, additional radial mixing transverse to the streamlines is necessary, which can be achieved through shearing, expanding, compressing, kneading, and utilization of backflow and spiral flow [15, 18]. Finally, turbulent mixing provides the greatest effectiveness. This is due to spatial and temporal flow fluctuations requiring the fluid particles to reorient continuously along Lagrangian trajectories [18], which leads to an effective turbulent mass transfer.

Mixing processes are generally described by statistical parameters such as mixture quality, mixing time, and residence time distribution. Furthermore, the dimensionless Reynolds number and the specific power input are important parameters in determining mixing efficiency. All these parameters (which are briefly summarized below) are also used for scaling-up and scaling-down of mixing processes.

3.2.1 Mixing Quality

In general, mixing quality is typically defined as the deviation of a measured property related to the average value. A number of indices are used to quantify mixing, which differ depending on the application [19–21]. Six specific mixing quality conditions are generally recognized: completely unmixed systems, ideally homogeneous mixing, demixing, homogeneous random mixing, real mixing, and texture mixing [20]. In a completely unmixed system, all components are locally separated (Fig. 3.1a). An ideally mixed system (also known as an ideally homogeneous system) is similar to a crystal lattice, in which every component x adjoins the same number of components y (Fig. 3.1b). As illustrated in Figure 3.1c, demixing is characterized by spatial separation of components commencing adjacent to the walls of the system. A homogenous random mixture (Fig. 3.1d) constitutes a condition in which there is identical probability that component x or y will be encountered after sufficient mixing time, which is to be expected as mixing is a random process. In practice, the actual mixing condition (Fig. 3.1e) in technical mixing systems lies between the conditions shown in Figure 3.1b,d. Texture mixing (Fig. 3.1f) has no importance in biomanufacturing.

Figure 3.1 Mixing conditions describing mixing quality: (a) completely unmixed system, (b) ideally homogenous mixing, (c) demixing, (d) homogeneous random mixing, (e) real mixing, and (f) texture mixing

(adapted from Reference 20).

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3.2.2 Mixing Time

Mixing time is the second parameter used to quantify mixing efficiency and represents the time necessary to reach a defined mixing quality. A degree of mixing of 95% is often assumed to represent an appropriate performance in industrial mixing systems. But higher demands also exist in special applications.

3.2.3 Residence Time Distribution

Residence time distribution, introduced by Danckwerts [22], describes the probability of elements to remain within a continuously operated apparatus for a certain time. Thus, spatial and temporal mixing can be evaluated [23].

3.2.4 Reynolds Number

The Reynolds number describing the ratio of inertial forces and viscous forces is named after the physicist Osborne Reynolds. It is used to analyze the fluid flow regime and is regarded as one of the most important parameters in model theory. For example, laminar flow occurs at low Reynolds numbers and is characterized by smooth and constant fluid flow. Turbulent flow develops at high Reynolds numbers. Unlike laminar flow in which viscous forces prevail, turbulent flow is characterized by inertial forces, which tend to produce flow instabilities, such as random eddies.

3.2.5 Specific Power Input

The specific power input, defined as the mass- or volume-related quantity of power introduced into a system by a drive mechanism, which can be mechanical, hydraulic, or pneumatic, is a further critical parameter used in optimizing the design of mixing systems, in determining their efficiency and also in scaling-up [24]. A high specific power input will usually result in short mixing times. However, the shear stress in such a mixture will typically