Freeze Bulk Bags: A Case Study in Disposables Implementation

Abstract

Bulk storage, freezing, and transfer are important steps which ensure that the final product is safely and promptly delivered to fill–finish sites and patients. Current bulk freeze-thaw practices use predominantly stainless steel systems. Unfortunately, stainless steel bulk freeze-thaw systems have their share of disadvantages. This article addresses Genentech's evaluation of single-use technologies for bulk freeze-thaw, storage, and transportation, including operational and functional testing, the mechanical properties of film, controlled freezing, and the risks involved in bulk shipping.

Although disposable bioprocess containers (BPCs) are increasingly being welcomed into biotech facilities, plant managers have not been as quick to introduce disposable bulk freeze-thaw applications. Bulk storage, freezing, and transfer are important steps which ensure that the final product is safely and promptly delivered to fill–finish sites and patients.

Nalgene, Inc.

As with other traditional manufacturing processes, current bulk freeze-thaw practices predominantly use stainless steel systems. Unfortunately, stainless steel bulk freeze-thaw systems have their share of disadvantages. From integrity testing and passivation challenges to continued upkeep and shipping validation, the number of man hours and support teams needed to sustain these stainless steel systems are high. However, as disposables move more into mainstream manufacturing, so too will disposable bulk freeze-thaw systems.

Storage Hold Process and Transporting Bulk

As it becomes compulsory to lengthen the lifetime of a protein product to reach patients worldwide, considerable effort and thought must go into the storage hold process. One option for bulk protein storage is holding the product in a liquid-state, either in a stainless steel tank or a disposable container. Although a viable option, the use of liquid-state storage has its own drawbacks. First, proteins may aggregate, resulting in product loss. Second, oxidation may cause some spontaneous reactions that would be detrimental to the bulk. Regardless of these concerns, there has been data that proves that this option may be suitable under certain conditions.¹

A less risky method for transporting bulk is through freezing, which slows down aggregation and oxidation to the point where they become less of a concern. Freezer systems used in the biopharmaceutical industry vary depending on the type and amount of product, but bulk traditionally is frozen in scalable containers ranging from small plastic bottles to large stainless steel tanks.

Sartorius-Stedim's CryoFin

One example of a stainless steel freeze-thaw system would be Sartorius-Stedim Biotech's CryoFin. CryoFin cryopreservation technology consists of several separate components that include CryoVessels, thermal control units, and mixers. The freeze-thaw is controlled by active and passive heat transfer surfaces and is specifically designed for large-scale freezing of biotherapeutics. Although this system is novel and offers valuable flexibility to both the manufacturers and clients, it has some drawbacks. For example, because of its need to be tested and validated before use for sterile hold and other sampling and validation qualifications (which could take months or even years), by the time a stainless steel system is commissioned, the particular campaign in which the system might have been useful may have already been completed. Furthermore, the amount of capital necessary to invest in such a system is significant compared to equivalent disposable systems.

Another option would be for bulk to be frozen and transported in disposable BPCs. An example of this application would be a container developed by Thermo Fisher Scientific. Using their HyQ CX5-14 film, Thermo Fisher Scientific's single-use bioprocess container system was developed and tested to satisfy certain important characteristics that all containers systems must require. Furthermore, integrity tests were done on these containers when they were frozen and transported while lying flat, in the hanging position, and during repeated freeze and thaw cycles. The test results indicated that all the units maintained fluid integrity and could be used as an alternative to stainless steel that negates the testing that stainless steel systems require.

Sartorius-Stedim's 6L Celsius flexible freeze and thaw clamshell

As with stainless steel, disposable bulk containers do have their own set of drawbacks. For example, BPCs and flexible tubing usually cannot handle more than a few psi of pressure. Temperature limits are also an important detail to consider when companies are looking for disposable systems or parts. Most disposable systems are validated for operation at standard biopharmaceutical temperatures, but some BPCs or their connectors and tubing may need additional validation for storage at low temperatures. For example, certain biotech raw materials such as media or cell lines must be stored at –80 °C, so it is necessary that commercial manufacturing facilities validate all single-use technologies to ensure compliance with these and other specific temperature regulations. Although it can be argued that stainless steel vessels are better than single-use systems because they do not have temperature limits, stainless steel vessels often contain gaskets and elastomers that have different heating and cooling properties, which can cause leaks and sterility problems.

Nalgene Nunc's bag management system

Operational and Functional Testing

Manufacturers of bulk shipping containers typically put their products through three different types of functional testing:

• shipping and transportation

• drop

• handling.

Shipping and transportation testing includes packaging bioprocess containers into a variety of shipping and support containers either individually or in groups depending on the type of unit being tested. Frequently, the units are filled with water or some equal buffer substance at the nominal volume of the container before package testing. The units then undergo trials of shock and vibration tests to simulate conditions stipulated by the International Safe Transit Association.² Next, a BPC endures individual drop testing to assess the durability of the unit without the exterior packaging. In this test, units are dropped from a height of one meter onto a hard concrete surface to simulate the worst-case scenario drop that might occur in normal shipping operations. Finally, handling tests ensure that the BPCs are operational from the beginning until the end of the manufacturing process. The units go through a cycle process several times for at least 48 h, in which they are frozen horizontally at –85 °C, and then incubated at +60 °C.

Mechanical Properties of Film

Along with functional testing, all BPCs must also undergo specific tests to ensure the compatibility and robustness of the films that will hold the bulk product. Among the specific properties that films are tested for and required to possess include (but are not limited to):

• tensile properties

• toughness

• elasticity

• puncture resistance

• tear resistance

• flex durability.

Tensile properties 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. This number is often recorded as the percent of film elongated compared to its original length. When a specimen has a high elongation at break, it can endure a large amount of deformation before breaking and is considered a highly flexible film.

A material's ability not to fracture when stressed is measured as a film's toughness. This describes a film's maximum absorption of energy before the film rips. Toughness is calculated by finding the area underneath a stress–strain curve, but toughness and strength do not necessarily mean the same thing. For example, a brittle film material would be one that is strong but not tough.

The elasticity of a film is measured by Young's Modulus (also known as an elastic or secant modulus). The more rigid a material is, the higher the elastic modulus is said to be. If the modulus is lower, then the film material is more flexible and easier to mold. This elastic modulus can be calculated by determining the slope of the line on a stress–strain curve from the origin of the graph to the strain is held constant at a value of 2%. This provides an estimate for the rigidity or flexibility of a material.³

The 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 resistant materials are able to absorb a lot of the energy of impact by increasing elongation or resistance to deformation.

Tear resistance is a combination of tests having some properties of an elastic modulus measurement and tensile strength. As the name implies, tear resistance is the study of how much a film is able to resist tear. There are many ways to evaluate tear resistance including loading the film materials at small rates. Another way to measure tear resistance would be to find the amount of force necessary to induce a slit across a piece of material. Though there are other standard methods available, the latter is the most commonly used method.

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. This test is dependent on how the film is to be used in the process because this determines the number of cycles a film must undergo during testing.

Finally, another important mechanical property that must be considered is the glass transition temperature (Tg) and the brittle temperature of film material. Tg is defined as the temperature at which a polymer goes from an inflexible, rigid state to an elastic, flexible state.⁴ This quality is very important for determining the suitability for a BPC. When BPCs are used as a freezing application, they must be able to withstand the extreme freeze temperatures that protein drug products must be stored at. Low Tg values do not necessarily mean good resistance at low temperatures. Because low Tg materials can absorb more energy from impact or loading forces, they are able to withstand longer and function better. Several films by vendor companies have been established to have a Tg of –22 °C while others have developed a film with a Tg of –31 °C. Both of these films, despite their different Tg values, have the same cold crack temperature definition of –80 °C thus making them fit for use in standard biotech freeze-thaw applications.

Controlled Freezing

Bulk freezing of macromolecules is a method that makes it safe to store and transport a drug product while reducing microbial contamination. The active pharmaceutical ingredient (API) is an important part of the bulk drug product and must be preserved for a protein therapeutic to work. The degradation of the API is detrimental to the bulk product and as such the rate at which the protein is frozen must be closely regulated because it can be both harmful and beneficial to the API.

Because the biological activity of a protein can change with the mere formation of a soluble aggregate, precipitation freezing must be closely controlled. If the freezing process is too slow, then solutes may form or the pH may shift because of buffer salt crystallization. The formation of solutes leads the API to become concentrated and the formulation composition changes, leading to the aggregation or precipitation of the protein product and chemical degradation of the API. If the rate of freezing is too rapid, small ice crystals may form with large surface areas. This phenomenon leads to a large interfacial area between the protein and ice crystals that also can lead to aggregation and precipitation of proteins. In either case, the result is an unstable protein product.

Many freeze studies were conducted at the Oceanside clinical plant to examine this process and to characterize the freezing process of a product formulation buffer in disposable bioprocess containers. The results of this experiment (Figure 1) not only proved the fluctuations and trends in freezing bulk in disposable bags, but also showed a variation of freeze rates in terms of where the product was placed inside the freezer. These freeze studies reinforce the fact that it is necessary to examine which freeze rates work best for any new system or product being introduced into a facility.

Figure 1. Trends and fluctuations in freezing bulk product in flexible bags, and variation in freezing rates depending on bag location in the freezer.

The rate at which the bulk drug product is frozen is only one of the factors that affect protein recovery. Container dimensions also play an important part in API recovery. The freeze distance is the space from the edge of a container to its center and thus it is not always accurate to use scaled-up or down models to describe API stability in bulk-freeze containers. To receive accurate data, studies must be conducted for each individual container used for a freeze-thaw process.

Thermodynamics of Freezing

The freezing of bulk drug product is a complicated process that can be divided into several segments. In the initial cooling phase, the product is chilled to the freezing point of the liquid. The initial drop of the freeze curve eventually plateaus off at this point. This plateau occurs because at this point, 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. Next, 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.

Another variable that must be considered is bulk-scale freeze concentration. Bulk-scale freeze concentration is a phenomenon that can happen if a solution is frozen in a way that allows for solutes to disseminate from the slowly growing ice surfaces, which can open a way for convection to occur in the unfrozen parts of the solution. This can be a major issue to deal with because it may lead to variances in a drug product's stability and formulation concentration. The freeze concentration can lead to changes in the protein and excipient concentrations in a solution, which means that certain parts of the solution will freeze faster or slower than others. Another issue that can be a problem when considering freeze concentration is scale-up because models on a small level may not be representative of what happens when container shape, size, and volume change.

The most important point that occurs on the freeze characteristic graph is called the last point to freeze (LPTF). LPTF is defined as the longest freezing time experienced by a product in a vessel in which the product is in contact with the liquid phase. The LPTF always occurs in the geometric center of the product when freezing because bulk product freezes from the outside in and from the bottom up. Because 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. As a result of a disposable freeze study at Genentech, freeze curves of disposable systems were shown to be very similar to curves found for stainless steel containers, thus proving the comparability of disposable bulk freeze-thaw containers with their stainless steel counterparts.

Inherent Risks of Bulk Shipping

The risk in using disposable freezing and shipping systems in bulk storage and transfer is obvious. Many biotechnology companies are reluctant to use disposables because they are novel and untried systems. Additionally, there are other issues regarding the use of disposable bulk freezing and bulk storage systems that are not present with stainless steel applications. Some of them include the following:

• leaching

• absorption of bulk product to the bag

• variation of the APIs because of storage in the bag (stability)

• light sensitivity

• integrity issues with manufacturing of the bag (e.g., connectors, ports, transport).

Testing for extractables or leachables is a requirement before implementing any disposable bioprocess container into a manufacturing facility. Leaching can be described as the removal of soluble or insoluble materials from the container that are released into a product solution during a process. Obviously, this can be an problem when dealing with bulk product and purified substances.

Because most storage systems are transparent or semitransparent, light sensitivity also can be an issue. Photosensitive drugs or proteins that are activated by light must be specially handled if they are to be stored in a BPC, which will allow in light. Also, the integrity of a BPC must always be visually inspected before use because anything that results in the loss or potential loss of a product is a high-risk investment. Seams tend to be the weakest points and may become brittle and subject to breakage at low temperatures. Also, all ports and tubing on the BPCs must be supported when frozen so that they are not liable to break. The increased dependency on vendors may be seen as a major disadvantage, but this is why it is always beneficial to have a validated backup vendor so that there will always be a continual supply to the manufacturing facility if problems arise.

Another challenge for implementing bulk storage systems is finding satisfactory exterior containers for storing the bulk, both in liquid and frozen state. A viable option that has already been developed at Genentech is 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 protocols when it comes 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 without the aid of dry ice and is also space efficient and disposable.

A further concern that must be taken into account is sampling. Because the bulk fill transfer step from the hold tank into the disposable BPCs is considered an intermediate step, samples must be taken from these bags. The sampling of each pack used can be a highly time-consuming process both on the technician side and for the quality assurance and quality control groups monitoring the samples. If there were fewer bags to manipulate, then there would be less manual manipulations and less sample errors because contact would be minimal. Sampling through a single port at one connection point to fill multiple sample bags would be ideal as taking samples one by one is not as precise and leaves much more room for manual manipulations or inaccuracies. Because sampling requirements will be specific to the product produced and the regulations for its production, a single solution to the industry's sampling needs has not yet been established.

Finally, as volumes increase with large-scale disposables, so do the shipping containers and pallet tanks that accompany them. One reason disposables are such an appealing option is that they are lighter and more mobile than their stainless steel counterparts, and therefore, it does not take many people to lift a single bag into a freezer or a lot of machinery for shipping.

Conclusions

As medicine becomes more personalized toward the patients, drugs must be developed faster and have lower failure during clinical trials. To satisfy both demands, it will become necessary to streamline processes so that multiple drug substances can be quickly and effectively manufactured and turned around. Therefore, disposable bioprocess containers represent not only a sound investment in a current process but also an investment capable of handling the quick changing pharmaceutical environment ahead.

Adam Goldstein is a senior manager, Jacqueline Loesch is a manufacturing scientist, Kellen Mazzarella is a process development engineer, pharma packing and engineering, Tim Matthews is process development engineer and group leader, and Grant Luchsinger and Diane S. Javier are associate engineers, all at clinical operations, Genentech Inc., Oceanside, CA, 760.231.2399, adamsg@gene.com Luchsinger and Javier were summer interns when this article was written.

References

1.Singh SK. Storage considerations as part of the formulation development program for biologics. American Pharm Rev. 2007;10:03/04.

2.International Safe Transit Association. Guidelines for selecting and using ISTA test procedures and projects. Available from: http://www.ista.org/forms/ISTAGuidelines.pdf.

3.Thermo Fisher Scientific. Characterizations of single-use bioprocess container systems based on HyQ CX5-14 Film. Available from: http://www.thermofisher.com/global/en/home.asp.

4.ER75272 recommended operating Temperature range HyQCX5-14 film. Michael Goodwin and Greg Elgan, Hyclone Laboratories, Inc. Engineering Department (Tg ISTA Testing).