Disposable Freeze Systems in the Pharmaceutical Industry

Roche Diagnostics GmbH
Genentech Inc.

A journey from current stainless steel to future disposable freeze systems in clinical and large scale manufacturing

Introduction

Recombinant DNA technology has enabled the industrial production of monoclonal antibodies and recombinant proteins for targeted treatments of several diseases like cancer, viral infections, and so on. Therefore, the production of biotherapeutics is of increasing importance for the pharmaceutical industry. Recently, a large number of monoclonal antibodies and therapeutic proteins have been approved and will be the major source of revenue for the industry in upcoming years [1-5]. The expression system of choice for the production of complex recombinant proteins and antibodies are mammalian cells cultivated in suspension [6-9]. Manufacturing scales up to 25 m3 operated in a batch, fed batch or repeated batch mode are state of the art in the industry. Following the cell culture process, a sequence of chromatography, filtration and concentration steps are performed and the purified bulk drug substance is frozen for improved long-term stability [10, 11] - a typical antibody production process is shown in Shukla and Thömmes 2010 [12].

However, the pharmaceutical industry is facing many challenges in upcoming years due to loss of patent protection, increased competition, increased R&D costs, decreased healthcare budget, etc. [5]. In order to overcome these challenges, the industry has introduced several initiatives along the value and supply chain to decrease costs, increase success rates and provide excellence in operations. Amongst others, supply chain resilience and manufacturing costs play a significant role in actively supporting the strategy of the company and contributing to a competitive advantage [13]. Along with six sigma and lean methodologies to avoid waste and decrease process variability in business and manufacturing processes, the successful management of technologies and (incremental) innovation from R&D to manufacturing is of increasing importance. The overall goal is to increase agility and flexibility while at the same time improving robustness and costs in the 21st century manufacturing organization [14-17].

Innovation and technology management models, as well as integrated development approaches, have been described in several industries and at various levels of organizations [18-23]. However in a strictly regulated environment, the introduction of mature technologies needs to follow the approach of demonstrating comparability in terms of product quality and applicability from a business perspective. Risk management, knowledge management, ongoing process validations, etc., are elementary processes for the successful implementation of mature technologies in new and existing manufacturing processes. A good example is the usage of disposable systems in the pharmaceutical industry, which is a well-established technology in most areas of today’s production processes. The technology evolved from disposable filtration membranes and bags for storage of simple solutions, to more advanced systems for mixing upstream and downstream solutions along with media, bioreactors, UFDF and membrane chromatography for downstream processing up to disposable facility solutions.

Disposable systems offer advantages such as increased flexibility, minimum pre- and post-use efforts, ongoing validation efforts, and so on. Several solutions have proven their efficiency and applicability in commercial and clinical manufacturing processes.

In recent years, disposable bulk drug substance storage containers for active pharmaceutical ingredients have become more important as they off er several advantages over existing standards in pilot and large scale. However, specific aspects need to be considered and evaluated during the implementation of this technology in order to mitigate business risks, operational handling perspectives, and most importantly quality and regulatory aspects. This article will provide guidance for the assessment of disposable freeze systems for clinical and upcoming large scale commercial solutions from several perspectives including detailed discussions around validation, operational handling, supply chain simplification and risk mitigation. Two case studies will be presented to illustrate these aspects in further detail.

Stainless Steel Tanks - Gold Standard for Bulk Freeze

Figure 1 - 300 L Stainless Steel Bulk Drug Substance Storage Vessel

Following the purification of the fermentation broth, the bulk drug substance (BDS) is stored in containers at temperatures ranging from liquid storage at 2-8°C to frozen storage down to -50°C. Liquid storage from 2-8°C can have deleterious eff ects on critical quality attributes and lead to degradation of the proteins over time. For this reason, most monoclonal antibodies (Mab) processes store the purified bulk drug substance frozen to limit protein degradation and elongate stability over time. Depending on the scale and the lifecycle of the product, the standard for many manufacturing practices currently employs the use of stainless containers or plastic bottles from clinical to full commercial (5 L – 300 L vessels). Traditionally bulks have been frozen in small plastic bottles or PFA carboys and as processes are scaled up, the requirements for final bulk volumes increased as well and larger stainless steel tanks became the norm for the industry. Stainless steel systems have provided a proven and established track record of capabilities for the industry. Since the freeze process of BDS is a critical step from a product quality perspective and a business perspective, a robust system and process is required for freezing, storage, and shipment.

Although stainless steel systems have become the “gold standard” of the industry, these systems inherently impose intensive resource, time and staff aspects including: maintenance, cleaning, validation, lifecycle management and logistical complexity. These aspects have increased the need for disposable solutions.

Stainless steel systems can have a considerable eff ect on supply chain operations – especially in a global environment with a grown asset base. With the engrained concept for lean and “just in time” manufacturing, supply chains need to be fl exible, reliable and efficient. Simplification is key to increase the performance of supply chains in terms of agility, robustness and cost eff ectiveness. The complexity of large scale BDS stainless steel containers can rise quickly. Next to the management of filled BDS, the empty tanks require basically a separate tank management strategy, which is resource and staff intensive. Below are some areas of concern:

  • Facing complexities of diff erent volumes, material qualities, filings, etc.
  • Coordinating the planning, tracking and scheduling
  • Maintaining and cleaning a large facility that stores empty freeze and thaw tanks
  • Aligning requalification and revalidation of the vessels with schedule
  • Confronting lead times and investment for the implementation of new freeze and thaw tanks
  • Managing space requirements, maintenance and operations costs of -20°C walk-in freezer
  • Ensuring safety with freeze and thaw tanks handling (size and weight)
  • Dealing with operational handling and costs
Figure 2 - Schematic supply chain for stainless steel large scale bulk drug substance tanks.

An exemplary global supply chain for a large manufacturing network for tank management, storage and shipment is shown in Figure 2. Figure 3 illustrates the complexity from an operational perspective at manufacturing site level.

Figure 3 - Operational steps at the diff erent sites for the stainless steel vessels. In process controls, validations and potential re-processing is not shown.

All of the described eff orts and issues make using and maintaining a large network of stainless steel tanks challenging, however there are new opportunities at full manufacturing scale volumes that are beginning to be off ered by disposable manufacturers. For clinical or commercial use at small volumes these have already been implemented and have proven efficacy [24-26].

Single-Use BDS containers

With the described complexity in the processing systems and the increased manufacturing capacities globally, there is a continued need for technical improvements in the freezing process and storage equipment for BDS. This need has many biotech companies turning from stainless steel containers to single-use disposable assemblies. Improved confidence in process capabilities, stability, robustness and the significant lower operational costs are driving the implementation of disposable assemblies in production [26].

Disposable bottles have been used for clinical and commercial supply before. However, this use still requires significant manual intervention and complex dispensing units, both imposing complexity in operating these systems as well as an open process step, which has a risk of contamination. The high value of biotech products at the final bulk drug substance step demands robust process throughout preparation, fill and freeze, shipping, and thawing. Therefore disposable bags off er advantages to established single-use bottles.

The application for so-called “disposable bags” or bioprocess containers varies from simple media containers, disposable sampling bags, all the way up to the storage of active pharmaceutical ingredient in the final BDS container. Implementation into clinical and commercial manufacturing has been reported successfully [24]. However, the system of choice depends on formulation characteristics, volumes, process requirements and requires careful assessment. A comparison of diff erent systems and assessment of their characteristics is shown in Table 1 (scalability, shipping robustness, etc.). In terms of scale, most disposable processes are not entirely scalable; however, there have been numerous advances for single-use technologies in commercial production. [27].

Table 1. Aspects for diff erent freeze systems and volumes. +=low, ++=medium, +++=high. Sc = Supply Chain; OR = Operational Robustness; Op. Eff . = Operational Eff orts; Val. = Validation

There are several advantages and drivers for the implementation of disposable bulk drug substance containers. Some benefits include:

  • Reduced risk of contamination due to closed system
  • Flexibility in volumes and configuration
  • Elimination of pre- and post-use steps and handling
  • Decreased supply chain complexity and one way logistics (see figure 4)
  • Reduced maintenance, ongoing validation eff orts
  • Energy and utility savings
  • Lower cost of quality due to reduced testing, lower risk for foreign objects, etc.
  • Lower upfront investment and lead times
  • Lower storage space compared to large scale steel vessels in the warehouse
  • Ergonomic and safety aspects
  • Low temperature product stability

As shown in Figure 4, a completely disposable supply chain achieves significant simplification in comparison to the exemplary supply chain for stainless steel tanks shown in Figure 2. Consumables will be delivered directly by the supplier to the drug substance-manufacturing site according to bill of materials. Inventory, cycle and safety stock can be managed by the vendor including supply chain risk mitigations. Waste disposal needs to be done according to good environmental practices and recycling options should be pursued.

Figure 4 - Supply chain for a full disposable bulk drug substance is reduced to shipping between drug substance and drug product sites. (VMI= Vendor managed inventory)

Most disadvantages are associated with the waste treatment, extensive and specific extractable/leachable studies, potential challenges of guaranteed supply, change management, limited scalability from clinical to full commercial, control of freeze kinetics, limited experience in commercial manufacturing, breakage of bags during transport, etc. Since these aspects might lead to a supply chain disruption, the risk and vulnerability of the supply chain needs to be managed carefully.

However, these challenges are not unique to stainless steel vessel, like for example leaking tanks, cleaning validation, etc. While assessing the suitability and implementation strategy, several critical aspects should be assessed carefully for the usage of disposable systems for bulk drug substance storage. Overall, the systems show substantial advantages compared to the stainless steel vessel, which can lead to significant improvements if managed right. This article will address some of these aspects.

Testing, Qualification and Validation for Single-Use Systems

Film Characteristics

In general, the bioprocessing film container usually has a multi-layer film composition which varies from vendor to vendor. The film is not composed of one type of monomer plastic. An example of such a composition is illustrated by a Sartorius Stedim Biotech Flexel 3D film structure shown in Figure 5.

Figure 5 - Composition of film material Validation Guide Flexel 2D and 3D Bags, Sartorius Stedim Biotech, 1 = Polyethylene Terephthalate (PET); 2 = Polyamide (PA); 3 = Tie Layer; 4 = Ethyline Vinyl Alcohol (EVOH); 5 = Ultra Low Density Polyethylene (ULDPE) – Medical grade fl uid contact layer

The outermost layer is a polyethylene terephthalate followed by a polyamide, ethylene vinyl alcohol, and finally an ultra-low density polyethylene layer. Each of these layers has a tie layer in between to bind them together. The multi-layer composition provides key performance when it comes to addressing issues due to gas and moisture exchange and physical strength for the bag components. Therefore, film characteristics for disposable bioprocessing containers must undergo rigorous testing. According to Barbaroux and Sette 2006, there are several requirements that are necessary for films to possess including (but not limited to) [28]:

  • Tensile properties
  • Toughness
  • Elastic or secant modulus at 2%
  • Puncture resistance
  • Tear resistance
  • Flex durability

Tensile properties are used to describe the ability of a film to stretch when stressed to its limit. A tensile test is described as the process of elongating a film sample and measuring the strength that results. The moment the film or object breaks is when elongation is recorded. Scientists often record this number as the percent of film elongated compared to its original length. When a sample has a high elongation at break, it is able to endure a large amount of deformation before breaking and is considered a highly fl exible film.

A film’s ability to resist fracture from applied stress is measured as its toughness. This describes its maximum absorption of energy before tearing. Toughness is calculated by measuring a stress curve. Toughness and strength are not necessarily interchangeable. A film material can also be considered brittle, meaning strong but not tough. This becomes critical during the freeze process where the film’s properties change due to the low temperatures it is subjected to.

A test that measures the durability of a film material while in use is called puncture resistance testing. Puncture resistance testing is comparable to tensile toughness because it assesses the strength of a film and its extensible properties. Good puncture resistance materials are able to absorb a lot of energy of impact by increasing elongation or resistance to deformation.

Tear resistance is a combination of testing, having some properties of an elasticity measurement and tensile strength. Tear resistance shows the films ability to resist tearing. There are many ways to evaluate tear resistance including loading the film at small increments as well as testing the amount of force necessary to induce a slit across a portion of the film.

Flex durability is tested using the Gelbo Flex test. The Gelbo Flex test pushes film material horizontally while simultaneously twisting and crushing the film. This is done several times and failure is marked by observing the amount of pinholes that were formed in the film material after the procedure [28].

Another important aspect to consider when evaluating a film for freezing is the glass transition temperature (Tg). The glass transition temperature (Tg) is defined as the temperature at which a polymer goes from an infl exible,rigid state to an elastic, fl exible state. This quality is very important for determining the suitability for a freeze container. When BPCs are used as a freezing application, they must be able to withstand the extreme freeze temperatures that protein drug therapeutic product must be stored at. Low Tg values do not necessarily mean good resistance at low temperatures; however, since low Tg materials can absorb more energy from impact or loading forces, they are able to withstand longer and function better. Tg measurement can vary according to test method, and several film manufacturing companies have been established to have a Tg of -22°C while another company has developed a film with a Tg of -31°C. Both of these films, despite their diff erent Tg values have the same cold crack temperature definition of -80°C. Cold crack temperature is a measure of a film's toughness at low temperatures and is determined as the lowest temperature at which a specified weight can be dropped onto a folded piece of the film without observing a crack in the film (ISO8570).

Validation of Leachables and Extractables

Testing to ensure that the bulk container film is neither additive nor adsorptive to the process is critical and mandated by regulatory agencies (ICH Q3B, FDA guidelines for container closure systems). Some of the required testing includes the following:

  • Leaching
  • Absorption of bulk product to the bag
  • Changing APIs due to storage in the bag stability
  • Light sensitivity
  • Integrity issues with manufacturing of bag

Testing for extractable or leachable material is a requirement before implementing any disposable bioprocessing container into any manufacturing facility. Leaching can be described as the removal of soluble or insoluble materials from the container and released into a product solution by process. Obviously, this can be an issue when dealing with GMP bulk product and purified substances. Because most storage systems are transparent or semi-transparent, light sensitivity can be an issue. Photosensitive drugs or protein therapeutics that are infl uenced by light must be specially handled if they will be stored in a bioprocessing container that allows in light.

While most vendors conduct contact layer testing for their specific plastic films, either the customer or a contracted testing lab must perform testing specific to the storage conditions, solutions and hold times that exist in the specific process. The test conditions should closely bracket the actual conditions that the BPC will encounter as temperature, the aggressiveness of the solution, and the organic content of the solution all aff ect the performance of the BPC and ultimately the proposed process into which it is being implemented. Since plastics can release soluble or insoluble materials into products, it is necessary to also conduct a technical assessment to determine the sources and amounts of any extractable chemicals.

An extractable testing program should be initiated to estimate the quantity of chemicals that can potentially migrate from plastics into process streams by using model test solutions that bracket the process solutions. This bracketed approach generates a worst-case estimate for the maximum amount of extractables that could be concentrated within the product and evaluate the risk associated with the worst-case extractability findings. Other factors that must be controlled include maximizing the surface-tovolume ratio of the test container-solution combination and storing the containers for the actual time that will be used in production.

A second very important area that must be investigated is leachable testing, which is often confused with extractable testing. Leachable testing is performed under the actual process conditions. Often leachable testing is performed concurrently during engineering or technical development runs at scale to confirm no loss of product from the container and to detect for minimally leached contaminants from the film within the drug product. The leachable testing can be conducted at small scale; however an engineered solution that allows for the scale down of the freeze and transportation conditions for a disposable bulk drug container must be carefully designed in this case. Often the leachable testing is performed after bulk fill and freeze / thaw on the drug product. Known and unknown species are identified via multiple gas chromatographies and mass spectrometry analysis other assays for potential contaminates leaching during the production or hold phase on the bioprocess container. In order to accelerate timelines from clinical manufacturer of a target molecule to commercial launch, it is beneficial to utilize the same product contact film early on in the development stage of the product life cycle if possible. This ensures that stability and leachable information can be leveraged at full scale when the molecule is licensed.

Finally, rigorous change management for material changes and guarantee of uninterrupted supply is necessary. A strategic supplier-relationship management is necessary to allow early planning for potential changes and mitigate risks. Once the specific material and bag is established in a regulatory filing, changes of film materials, vendor manufacturing operations, raw material supply, etc., can have a severe impact on supply situation since there could be an impact to product quality and a critical supply chain disruption.

Secondary Containments Shipment Studies

The risk for use of bulk storage in disposable freezing and shipping systems appears to be obvious at first glance. The potential breach of a bag leading to potential contamination risk and loss of product is a bigger concern as compared to steel tanks. Annealing bag joints with glue and the presence of ports and tubing assemblies that may be sensitive to pressure and shock are also an area of concern and require special attention during shipment of frozen bulk. Therefore, secondary and tertiary containment are used for protection of the high value goods. In most cases, the bag is placed into the secondary containment before filling and freezing. Thermo-HyClone Inc. for example has an intermediate shipper that is manufactured according to Good Manufacturing Practices and can be frozen along with the bulk. Because the shipping dunnage is already in place as the product is frozen, there are less mistakes and the protocols can be simplified with regards to transporting the bulk to fill and finish facilities. The intermediate shipper with the bulk frozen inside is placed into an external shipper which can maintain temperature with the aid of dry ice. This external shipping system is also space efficient and disposable (see Figure 6). Some challenges to shipping include the primary container that holds the bulk and the external shipping system that the disposable bulk container is housed in. These challenges are identified below:

  • Designing the shipping container to fit the bulk and its attachments snugly
  • Robust temperature monitoring and last point of freeze control
  • Additional heat transfer barriers introduced with the secondary containment
  • Designing an external shipper that can maintain temperature
  • Movement of the external shipper in a safe and robust manner
  • Development of a shipper that is disposable and green to enable one way logistics

Since transportation robustness, safety and compliance is a critical step, manufacturers of BPCs typically put their products through functional testing according to transportation authority standards. For example ISTA has two types of tests:

  • Performance Tests result in a Pass/Fail assessment and are used to determine the viability of a packaged-product to survive normal shipment.
  • Development Tests compare relative performance of two or more designs or the same design from different suppliers.

The containers are generally tested for shipping/transportation, drop testing, and handling within the testing. Shipping and transportation testing includes packaging bioprocessing container units either individually or in groups into a variety of shipping and support containers depending on the type of unit being tested. Frequently the units are filled with water or some representative buffer substance at the nominal volume of the container before packaging testing. The units then undergo trials for shock and vibration tests to simulate conditions during transport handling and storage. Trial temperatures for most units range from -70°C to +23°C.

Figure 6 - Example for shipping containers. A) 20L Hyclone BDS Disposable container and disposable shipper B: Disposable Celsius LFT 100L container by Sartorius Stedim Biotech C) Disposable Celsius FFTp 6L and 12L containers by Sartorius Stedim Biotech

In order to mitigate the risks and provide sufficient support to the bags, a secondary containment – in most cases a hard shell – is used to protect the bag and tubing components during handling, storage and shipment.

Controlled Freezing

Independent of the storage container, the freezing process and the associated control is of major importance to avoid impact to product quality attributes (aggregation, cryoprecipitation). The freezing process can expose proteins to other stresses as a consequence of the removal of water as ice. The resulting cryoconcentration and desiccation of protein can cause osmotic stresses, precipitation, etc. Other freeze effects to proteins include ice interface formation, pH changes, and phase separation that can cause gradients in pH, salinity, protein concentration and permanent or reversible denaturation. Protein structure changes that occur as a consequence of such stresses have a greater probability of being irreversible, and are classified as freeze denaturation like protein aggregation, oxidation, etc. [29,30]. The control of the freezing process, design of freezing devices and the peripheral equipment realizing heat transfer are therefore of key importance.

Stainless steel systems have proven efficacy for controlled freezing to protect the product, minimize potency loss and allow for long-term stability without affecting the quality attributes [31]. At scale examples include the Sartorius Stedim Biotech freeze and thaw tanks. The stainless steel freeze-thaw system called the CryoFin™ cryopreservation technology consists of several separate components which include freeze and thaw tanks, thermal control units and mixers. The freeze-thaw is controlled by an active and passive heat transfer surfaces in the freeze and thaw tanks. The system is specifically designed for large scale controlled rate freezing of biotherapeutics. The prerequisites and requirements also need to be met by disposable systems. In freeze and thaw tanks, the cooled silicone oil is circulated through pipes as well as coils to increase the surface to volume ratio in order to enhance heat transfer.

In contrast, disposable bags have additional resistance layers because the actual product is not in contact with a conductive chilled metal surface. This can mean a decrease in heat exchange and potentially lower freeze rate, which need to be compensated. In the case of disposable systems shown in Figure 6, the heat transfer must go through the bag film, air, secondary shell, air and potentially other materials which cause inefficiencies. For smaller stand-alone bag systems (2-20L fills) placement into standalone freezers have successfully proven to cool via cold air convection. For the larger scale system Celsius® LFT two fins are slid into sleeves in the secondary containment holder box (Figure 10). The bag is housed into the secondary compartment and protected from the freeze fins by a thin plastic wall. The filled bag is frozen by circulating silicone oil through these fins – comparable to the technology used with freeze and thaw tanks. However, the additional heat transfer resistance compared to the stainless steel vessel needs to be overcome as more material layers are used for this system.

The duration, linear scale up and freeze rate of a molecule many times is individually developed. However, the consistency of the freezing process is critical and is required so that critical parameters can be monitored and assessed for consistency.

The rate at which bulk drug product is frozen is one of many factors that may affect the recovery of proteins. Container dimensions also play an important part in recovery of APIs. The freeze distance that the ice front must move across is one of the critical parameters to control. The freeze distance is the distance from the edge of a container to its center. Scale down models to describe API stability in bulk freeze containers must be appropriately designed to receive accurate data. Another important concept is that of freeze front velocity (FFV). The freeze front velocity measures the rate at which freezing occurs in a container. FFV is calculated by timing how long it takes a container’s content to freeze over the freeze distance.

The freezing of bulk drug product can be divided into several segments. At the beginning, there is an initial cooling phase where the product is chilled to the freezing point of the liquid. The initial drop of the freeze curve eventually plateaus off at the freezing point of the liquid. This plateau occurs because at this point in the freeze transformation, the liquid is in the middle of its liquid-solid phase transition state. The liquid-solid phase transition state is completed when the liquid’s latent heat of freezing is overcome. The third phase of a freeze curve occurs when the frozen solution is cooled down from the freezing point to the set point of the system after the latent heat of freezing has been overcome. These three phases are not only seen using the traditional stainless steel systems, but also in disposable bioprocessing freeze containers. The diff erent phases are shown in Figure 7.

Figure 7 - Diff erent phases during the freezing process [32]

A critical point to monitor during the freeze process is called the Last Point to Freeze (LPTF). LPTF is defined as the longest freezing time experience by a product within a vessel in which the product is in contact with the liquid phase. The LPTF usually occurs in the geometric center of the product when frozen because bulk product freezes from the outsides in and from the bottom up. Since the LPTF is representative of the worst-case scenario for protein stability, it can be used to correlate freeze results between stainless steel vessels and smaller scale systems. [32].

Operational Handling Aspects

Sampling

Additional challenges with Bulk Drug Substance (BDS) in large volume disposable containers deal with the logistics in obtaining a homogeneous sample from the disposable system. Mixing and sampling is a critical step in the bulk fills, freeze and thaw process. Samples are required during processing for several tests, including microbial testing, identity, concentration, etc., at drug substance and drug product sites. With the implementation of larger fill volumes in larger disposable bags vs. multiple smaller bag fills, there are fewer manual manipulations and fewer samples to be processed and therefore this reduces the chances of contamination. This can impact bag design or increase operational by welding, for example sample bags to existing tubing.

Pressure

The operating pressures used in the manufacturing process can limit the use of disposable systems in certain applications. This is why commercial manufacturing companies need to choose their disposable systems carefully. Bioprocessing containers and fl exible tubing cannot stand up to more than a few pounds of pressure.

Temperatures Sensitivities

Temperature limits are also an important detail to consider when implementing disposables. Most disposable systems are validated for operation at standard biopharmaceutical temperatures, but some bioprocessing containers and or their connectors and tubing may need additional testing and validation for storage at lower temperatures (such as -55°C or lower). Biopharmaceutical drugs are often held and stored at temperatures around -20°C or lower. There are a select number of products that must be stored at lower temperatures so it may be necessary to validate those technologies employed to ensure compliance with the lower temperatures. Stainless steel vessels are more compliant at lower temperatures than single-use systems due to their ability to handle lower temperature limits. However, stainless steel systems often contain separate components such as elastomers, gaskets or valves that are not compliant at these lower temperatures. Manufacturers of bioprocessing containers have designed and tested their materials to ensure that their products will remain intact and are compatible in a variety of conditions. Normally, testing conditions are simulated to evaluate how well the bioprocessing containers would perform under normal conditions and for its intended applications. However, weaknesses do not necessarily exist with the film material itself; in most cases the sample ports, welding of the bag and the tubing are the most sensitive areas and need to be protected during freeze, storage and shipment.

Mixing

In order to achieve a homogeneous solution before compounding, sampling, etc., the solution in the bag needs to be mixed adequately. Options like shaking, circulating the liquid by pumping, etc. are currently diploid. Mixing validations need to be performed to demonstrate suffi cient mixing capabilities. However, in some cases this imposes challenges for back design and secondary containment.

Other Aspects

Several other aspects need to be evaluated to assess the suitability of disposable freeze systems. Reviews are especially necessary in plant storage and movement, safety aspects, fit for existing equipment, and footprint suitability. These are more general product / facility fit aspects which will not be explored in this article.

As outlined above, several variables have to be considered for a reliable and robust implementation of disposable freeze systems. The following case studies demonstrate the evaluation for some of these aspects.

Case Study 1 – Implementation of a 20L Disposable Freeze System in Clinical Manufacturing

  Figure 8 - Hyclone 20L bag and external Clam Shell set up with dedicated -70C freezer

The main drivers for implementing disposable bulk freeze containers for this particular plant were acceleration of timelines and flexibility in bulk fill volumes. In the traditional start up for a biologics site, many different container sizes and configurations along with the associated freezers would need to be procured, tested and validated. The current facility as designed did not have dedicated freezer rooms, any stainless steel bulk cans, nor the large washer systems to adequately clean or CIP potential stainless steel bulk tanks which would require significant capital investment and additional lead times. A 20L disposable system was therefore tested. The main success criteria of the study can be summarized as follows:

  • Feasibility/capability for bag and clam shell system to reproducibly freeze without cracks, fissures, stretching to film or damage to the external shell.
  • Maintain bag integrity as measured by no breaches or leaks pre and post freeze thaw.
  • Smooth reproducible freeze curves showing transitions as expected from both existing processes in operations using steel as well as data published supporting controlled freeze studies exhibiting the phases shown in Figure 7.
  • Freeze system/chamber temperatures capable of freezing the maximum and minimum fill volumes reproducibly (7 L and 16 L).

As a first test for the bag freeze process, the standard formulation buff er was employed in order to characterize the freeze process. For the freezing process, the 20 L bag (HyClone Type CX514 ULDPE) was placed into the self-designed secondary containment container (Nunc Nalgene clam shell). The bag in the clamshell was then placed for simple yet controlled freezing in a standalone freezer. The freezer was set up with a pre-programmed ramp. The freezer temperature was controlled and alarmed via local control and monitored. The principle set up is depicted in Figure 8. Each bag was filled with formulation buff er (confidential) and then placed into the Revco freezer (Model #ULT3286). The following load configurations were tested:

All bags used in the study were monitored for temperature during each run. Through the course of the run, both the five refrigerator shelf temperatures as well as the internal contents of the bags were monitored by placing thermocouple probes into each of the bags.

Figure 9 - Exemplary freeze curve for a filling volume of 7 L.

The results for freeze studies are shown in Table 2 and an exemplary freeze curve for 7 L is shown in Figure 9. The results demonstrate that in all cases, the temperature of -36°C was obtained within the test criteria of less than 72 hours. From the freezing curves, the typical freezing profile for super cooling, nucleation and freezing point depression can be seen. The average time to reach target temperature for the 16 L studies was around 44.2 hours, with a minimum of 38.5 hours and a maximum of 47.3 hours. The average time to reach target temperature for the 7 L studies was 22.5 hours with a maximum of 25.6 hours. The bottom shelf was frozen first, followed by the fourth and top shelf indicating improvement opportunities for the air circulation. No irregularities on the bag surface or cracks or leaks after thawing were observed. A consistent and homogeneous freezing can be stated. Freeze curves exhibited nice smooth transitions with no major interruptions in the slope of the freeze profiles. Total time for the solutions to reach -36°C were all acceptable and exceeded the freeze times seen by traditional 35 L freeze tanks used in clinical operations.

Table 2. Results for the diff erent volumes tested in the bags to reach a temperature of -36°C.

During the experiments, the clam shells in the course of the study encountered some separation of the top and bottom shells after freezing which means a risk for further operational handling. However, the use of additional clamps was successfully implemented for additional runs. No leaks, breaches or other integrity issues occurred during the test runs. Drop test and shipping studies were performed with the set up shown in Figure 6 (A) which met all acceptance criteria.

For the implementation of the first freeze operations in clinical operations, two recombinant monoclonal antibodies were tested for stability and impacts via disposable bag freeze systems. Two bag films (EVA - Ethylenevinyl acetate and ULDPE – Ultra low density polyethylene) with the same volumes and customized bag utilities adapted to the clam shell were tested to mitigate potential supply chain risk. Along with the freeze curves outlined above, additional testing was performed for the product containing bulk drug substance. The results are summarized below:

  • All freeze curves could be reproduced
  • In all cases, no quantifiable leachables obtained from testing performed.
  • Product stability showed no impact to product quality compared to stainless steel or Tefl on bottles

              .    No impact to protein concentration or aggregation levels

  • No interactions with film by drug substance observed
  • No change of buff er composition (pH, osmolality, etc.)
  • No significant impact by potential cryoprecipitation

              .    Variables measured were IEC, SEC, protein concentration; Tween Content, Osmolality, pH, Color, Opalescence, and Clarity.

              .    Relevant inhomogeneity due to cryopreservation was not seen (pH, osmolality, etc.).

Overall the system was assessed to deliver comparable results and increased efficiencies without any impact on product quality attributes. During this case study, a disposable freeze system and process was successfully implemented in a clinical manufacturing environment. Other reported similar results for comparable systems [24]. Operational procedures describing set up, validation, sampling, mixing, etc., were implemented for routine operations.

Case Study 2 – Assessment of a Large Scale Commercial Disposable Freeze System

Figure 10 - Pictures of the Sartorius Stedim Biotech Celsius LFT Prototype.

Current manufacturing processes at full scale utilize resource and cost intensive stainless steel freeze and thaw tanks. These vessels are dedicated to specific products and have several disadvantages from an operational, supply chain and cost perspective as outlined above. A disposable system developed by Sartorius Stedim Biotech was assessed as a potential replacement for the large scale stainless steel tanks. The system consists of a 100L bag-in-box container with the equipment needed to freeze, thaw, transport, and store these containers. A 100L pre-sterilized single-use bag is placed inside a molded plastic box which serves as secondary containment; see Figure 10. The bag is filled with product and then frozen by lowering heat exchange plates into molded plastic sleeves which contact the main surfaces of the bag. This arrangement provides efficient heat transfer as well as protection of the bag. The progress of the freeze can be monitored by a temperature sensor located at the centerline of the container. The freeze/thaw unit, which includes a control system, uses an external chiller to regulate the temperature of the heat exchange plates; see Figure 10 and 6B. Up to three 100L modules may be operated in parallel to give a capacity range of 25L to 300L in a single system.

In order to assess the maturity of the system, the previously discussed aspects and system goals were assessed and compared to the established 300 L stainless steel vessel using a systems engineering approach. The five major success criteria and examples for assessment are shown in Table 3.

Table 3. Exemplary success criteria.

In summary, the available system offers several advantages in multiple categories. Outlines of some of the major advantages identified are listed below:

  • Reduction in cleaning
  • No reprocessing (rouge, debris, scratches, repairs, etc.)
  • Decreased risk of contamination and ability to maintain aseptic process
  • Reduced invests and lead times
  • Lower operational costs
  • Reduced supply chain complexity and one way logistics
  • High fl exibility of freeze volume 25 L to 300 L without additional equipment

Experimental Design / Set Up

An initial set of eight freeze operations were conducted in order to determine the freezing performance and reproducibility of the system. For each freeze, 101L of a solution composed of 50mM sodium citrate buff er at pH 6.3 and 10 wt. % sucrose were filled into a Celsius LFT container. The container was frozen using a prototype freeze/thaw unit using a temperature set point of -60°C. The temperature at the thermowell was monitored during the freeze process. Results are summarized in Figure 11 and Table 4. Each curve consists of three main segments. During the first segment, which lasts approximately 2 hours, the liquid product is cooled from the initial temperature to the freezing point of water. The second segment, which lasts approximately 8 hours, is marked by a gradual decrease in temperature as the water freezes, thereby concentrating the solute and depressing the freezing point. During the third segment, which lasts approximately 2 hours, the material is fully frozen and cooled to the final temperature. The average time from +10°C to -30°C was 12.3h; this compares favorably with a theoretical value of 11.6h calculated for the container geometry per [33].

Figure 11 - Celsius LFT Freeze Curve Data at Full Scale (Data Sartorius Stedim Biotech, Goettingen, Germany)
Table 4. Results from large scale experimental runs (Data Sartorius Stedim Biotech, Goettingen, Germany)

Data provided by the vendor for preliminary freeze curves indicate behavior comparable with the platform buff er. In order to address other points such as thaw kinetics, stability, and robustness during transportation, a detailed experimental plan has been designed and is currently in execution. Cryopreservation will be evaluated by taking samples at diff erent positions in the bag. The design seems to be feasible for safe transportation; preliminary data presented by the vendor for studies according to ASTM D 4169 and ISTA 7D standards showed promising results.

The Sartorius Stedim Biotech Celsius LFT system as tested performed well and can adequately freeze bulk material utilizing single-use bags in a consistent and controllable fashion. Additionally, the times required to freeze 100L of bulk solution are comparable to the results obtained in both stainless steel vessels as well as the scaled down freeze bags at 20L. All curves for the 100L scale were consistent and followed the expected freeze curve profiles and were devoid of pauses or unexpected anomalies. The data obtained so far indicates the saleable capability of controlled freezes without a systemic issue for breaking of bags during transport or freezing. Overall the first available system seems to show sufficient robustness for future commercial applications as assessed above. However, some aspects will need to be evaluated in more detail such as mixing, transportation and long-term stability of the bulk drug.

Future experiments will look at detailed freeze & thaw curves for protein solutions, mixing and sampling behaviors, and detailed risk assessments for end-to-end handling of the system. In particular, cryopreservation with diff erent filling volume to surface area ratios needs to be evaluated carefully. Protein-specific studies will include leachable/extractable analysis, product stability, etc. Logistic and supply chain aspects will be further studied to assess the full benefit of the system. These results will be published in a future article.

Conclusions

Disposable systems are a well-established technology in the pharmaceutical industry. Next to disposable filters, UF/DF membranes, bioreactors, etc., bulk drug substance containers for active pharmaceutical ingredients are making their way into clinical and commercial manufacturing. The disposable / single use system off er several advantages compared to the established stainless steel technologies. These can be summarized as follows:

  • Flexibility increase and risk reduction
  • Decrease of ongoing validation eff orts
  • One way logistics
  • Closed processing and reduced contamination risk
  • Less infrastructure and peripheral equipment required
  • Less resource and staff intense

However, the use and fit of the technology has to be assessed individually. Various critical aspects have been discussed in this article including validation, freezing, film properties, shipping, supply chain aspects, etc.

Two case studies show the exemplary assessment of these aspects and the suitability, advantages and challenges associated with disposable system for the storage and shipment of frozen bulk drug substance. Next to a successful clinical scale implementation a large scale solution was assessed in this paper and can be summarized as follows:

  • System requirements as outlined were met
  • Consistent and comparable freezing behavior/ kinetics to existing steel tanks
  • Robust shipping and reduction of supply chain complexity
  • Closed processing and reduced contamination risk reduction
  • Less infrastructure and peripheral equipment required (invest)
  • Less resource, staff and time intense

While the clinical system is already implemented, the experimental design for the large scale testing is still ongoing. Results so far indicate a viable commercial alternative to freeze and thaw tanks. Therefore we conclude that with the right regulatory, quality, supply chain and technology management strategy the implementation of a full scale disposable freeze system is possible and advantageous for the storage and transport of frozen bulk drug substance. Further experiments are ongoing and will be presented in a future article.

Acknowledgements

Jonathan Cutting and Mike Marciniak of Sartorius Stedim Biotech are acknowledged for supporting the project, experiments conducted and review of case study 2.

References

  1. Reichert J.M., 2001, “Monoclonal Antibodies in the Clinic,” Nat. Biotechnology, 19:819– 822.
  2. Reichert 2002 - Reichert JM. Therapeutic monoclonal antibodies: Trends in development and approval in the US. Curr Opin Mol Ther. 2002; 4:110–118.
  3. Reichert J.M., Pavolu A., 2004, “Monoclonal Antibodies Market,” Nature Reviews Drug Discovery, 3:383–384.
  4. Drapeau M., Sullivan F., Moniz Carpenter J., 2007, “Special Report: Blockbuster Then and Now-Trends for Billion-Dollar Drugs. Spectrum therapy markets and emerging technologies,” 12:1-39.
  5. Munos B., 2009, “Lessons from 60 Years of Pharmaceutical Innovation,” Nature Reviews, pp. 959-968.
  6. Xie L., Wang D.I.C., 1997, “Integrated Approaches to the Design of Media and Feeding Strategies for Fed Batch Cultures of Animal Cells,” Trends Biotechnology, 15(3):109—13.
  7. Qi H.N., Goudar C.T., Michaels J.D., Henzler H.J., Jovanovic G.N., Konstantinov K.B., 2003, “Experimental and Theoretical analysis of Tubular Membrane,” Biotechnology Progress,19(4):1183-1189.
  8. Wurm F.M., 2004, “Production of Recombinant Protein Therapeutics in Cultivated Mammalian Cells,” Nat. Biotechnology, 22:1393-1398.
  9. Sethuramann N., Stadheim T.A., 2006, “Challenges in Therapeutic Glycoprotein Production,” Current Opinions in Biotechnology,17(4):341-346.
  10. Heath C., Kiss R., 2007 Cell Culture Process Development: Advances in Process Engineering. Biotechnology Progress, 23: 46-51.
  11. Su W.W., 2003, “Bioreactors, Perfusion,” Encyclopedia of Cell Technology, 978 – 993. John Wiley & Sons. Inc.
  12. Shukla A., Thömmes J., 2010, “Recent Advances in Large-Scale Production of Monoclonal Antibodies and Related Proteins,” Trends in Biotechnology, 28:253 – 261.
  13. Wheelwright S.C., Hayes R. H., 1985 (January/February), “Competing through Manufacturing,” Harvard Business Review, pp. 2-12. Harvard University, Boston, MA, USA
  14. Bolten B. M., 2009, September, “Rethinking Pharmaceutical R&D: Will New Strategies Yield a Pipeline Payoff?” Spectrum Discovery and Innovation: Technologies, Strategies, and Dealmaking, pp. 1-22.
  15. Correa H. L., 200,1 “Agile Manufacturing as the 21st Century for Improving Manufacturing Competitiveness,” in Gunasekaran, A., Agile Manufacturing as the 21st Century for Competitive Strategy, 1st Edition (pp. 3-24). Kidlington, Oxford, UK: Elsevier Science Ltd.
  16. Cremer P., Loesch M., & Schrader U., 2009, April, “Maximizing Efficiency in Pharma Operations,” The McKinsey Quarterly - Operations Practice, pp. 2-4.
  17. D´souza A., Keeling D. J., Phillips R.D., 2007, September, “Improving Quality in Pharma Manufacturing,” The McKinsey Quarterly - Health Care.
  18. Schrage D.P., Mavris D.N., 2001, “Integrated Product-Process Development (IPPD) Through Robust Design Simulation (RDS),” in Gunasekaran, A. Agile Manufacturing: The 21st Century Competitive Strategy, 1st Edition, pp. 95-112, Kidlington, Oxford, UK: Elsevier Ltd.
  19. Sauber T., Tschirky H., 2005, “Structured Creativity – Formulating an Innovation Strategy”, Palgrave Macmillan.
  20. Burt J. A., 1996, February, “Department of Defense USA; Guide to Integrated Product and Process Development,” Version 1.0, Office under the secretary of defense, Washington DC, USA, 20301
  21. Biolos J., 1996, November, “Managing the Process of Innovation,” Harvard Management Update, Harvard University, Boston, MA, USA.
  22. Pisano G., 1998, October, Eli Lilly and Company: “Manufacturing Process Technology Strategy,” Teaching Note 9-692-056. Harvard Business School, Harvard University, Boston, MA, USA .
  23. Tschirky H., 2003, “The Concept of Integrated Technology and Innovation Management,” in Tschirky H., Jung, H.H. & Savioz P. (Editor), Technology and Innovation – Management on the Move – From Managing Technology to Managing Innovation-driven Enterprises. Zurich Orell Fuessli.
  24. Bieger, Boris, 2012, “Development and Implementation of Disposable Bags with a Freeze/Thaw-unit for Fast and Controlled Freezing/Thawing of Drug Substance,” 8th BioProcess Int. Conference Prague 18th - 19th April 2012.
  25. Samavedam, R., Goldstein, A. and Schieche, D., 2006, “Implementation of Disposables: Validation and Other Considerations,” American Pharmaceutical Review 9 (5): 46–51.
  26. Goldstein A., Schieche D., Harter J., Samavedam R., Wilkinson L., Manocchi A., 2005, October, “Methods and Considerations for Disposable Implementation,” BioProcess International, pp. 20-27.
  27. Pora H., Rawlings B., 2009, “A User's Checklist for Introducing Singleuse Components into Process Systems,” BioProcess International 7 (14): 9–16.
  28. Barbaroux M., Sett A., 2006, “Properties of Materials used in Singleuse Flexible Containers: Requirements and Analysis.” http://www. biopharminternational.com/biopharm/article/articleDetail.jsp?id=423 541&pageID=1&sk=&date=
  29. Franks F., 1995, “Protein Destabilization at Low Temperatures,” Adv. Protein Chem. 46, 105–139.
  30. Webb S.D., Webb J.N., Hughes T.G., Sesin D.F., Kincaid A.C., 2002, “Freezing Bulk-scale Biopharmaceuticals using Common Techniques — and the Magnitude of Freeze-concentration,” BioPharm International, 15(5):22-34.
  31. Nicolas Voute, Todd Dooley, Gaël Péron, and Eric Lee, “Disposable Technology for Controlled Freeze–Thaw of Biopharmaceuticals at Manufacturing Scale,” BioProcess International, Oct 2004:40-43.
  32. Singh. S, Kolhe. P, 2009, October, “Large-scale Freezing of Biologics,” BioProcess International, pp. 32-44
  33. Pham Q.T., 1984, November, “Extension to Planck’s Equation for Predicting Freezing Times for Foodstuffs of Simple Shapes,” International Journal of Refrigeration, Volume 7, Number 6, pp. 377-383.
  • <<
  • >>

Join the Discussion