Increasing Bioprocessing Efficiency, Single Use Technologies

The medical industry was the first to understand the benefits of using disposable devices, such as needles and syringes, to prevent risks of cross contamination. The technology was then extended to blood transfusion activities, and it was only 10–15 years ago that the biopharmaceutical industry started to use disposables. Initially, most of the applications were limited to storage, involving bags, tubing and filter capsules. Since then, significant progress has been made in the polymer and plastics industry; in particular, a number of organic polymers have been developed that are resistant to gamma irradiation, autoclaving and even sterilization-in-place, rendering the technology attractive and usable by the biopharmaceutical industry. Now, the industry is moving beyond storage-focused disposable technologies to more complex processing applications.

Single-use considerations

The adoption of single-use technologies has considerably increased with the emergence of a strong biotech sector. Disposable technology is seen as a boon to this industry because of the potential speed of implementation; the relative ease of the facility design; the reduction in validation requirements; and the decrease in capital investment and footprint. The idea is to adopt disposable bioprocessing operations very early in process development, which can be scaled up to large volumes. The scale at which disposable technology can be used has been questioned for a long time, but now the availability of bags, filters and devices that can handle thousands of litres is setting new capacity limits.

Suppliers are helping to drive the success of the disposables paradigm by keeping construction materials consistent across product and scale ranges. This reduces revalidation requirements and simplifies scale-up. The result is that disposable technology is now seen as a valid possibility from the initial manufacturing of clinical batches to full production scale, and is poised to be the technology of choice for all specific patient therapies.

Market drivers

Disposable technologies play a key role in minimizing any risk of cross contamination, which has been a strong driver for adoption by contract manufacturing organizations (CMOs). However, the time and cost benefits associated with the elimination of steam sterilization validation, cleaning and cleaning validation, as well as the reduction in the number of necessary connections may be seen as the strongest benefits for the broader pharmaceutical industry.

Cleaning validation is seen as a major hurdle in the biopharmaceutical industry. In fact, a high proportion of warning letters issued by FDA contain at least one remark pertaining to cleaning or cleaning validation (e.g., in 2002, this was close to 50% of cases).

Despite the relative ease of use of disposables, adopting and implementing this processing model requires careful evaluation. Table 1 is designed to help biotech/biopharm companies evaluate both process and cost models, as well as weigh the advantages/disadvantages and potential risks of adopting this model. Successful implementation also requires a high degree of commitment from management, quality assurance and processing teams. Some additional considerations to note include selection of the right materials and components, assembly design, developing a solid relationship with suppliers, training users, and developing an understanding of quality requirements.

Table 1. Comparison of hard pipe versus disposable factory.

Design and component selection

For a given bioprocessing unit, materials and components must be carefully selected. Product compatibility must be evaluated, and process parameters such as temperature and pressure along with sterilization methods need to be clearly defined. It is also important to look at in process measurements so that the appropriate sensor technology is chosen.

Criteria for establishing proper disposable system ergonomics include:

• Footprint and location of installation.

• Tubing — length, quality, attachment to the equipment through cable ties or improved methods.

• Elements to be integrated — for example, bags, filters and chromatography units.

• Connections — how to minimize the number of sterile connections, connection and disconnection capabilities.

• Sampling requirements during the process.

Validating and testing

Despite the fact that single-use systems significantly reduce validation requirements, they still need testing and validating. Initial testing involves checking the suitability of all components for the application and their relative position in the system. For example, operators should confirm that cable ties, where used, are in the right position and that the general assembly is strong enough to withstand handling and operating requirements. The entire system should also be tested for leaks.

Care should also be taken with packaging. Risk of damage during the irradiation and shipping processes must be carefully assessed too. Shipment studies can be conducted to ensure safe delivery at the user site.

Once packaging has been defined, and assuming that gamma irradiation is chosen as the sterilization method, it will be necessary to validate the irradiation procedure. The minimum required irradiation dose is 25 Kgy, and for most components the maximum is 45 Kgy. Dose mapping of the system is necessary to ensure that irradiation is falling within the requested specifications. Shelf life of the total assembly is also an important consideration when using irradiation as a sterilization technique as it will have a significant impact on inventory management at the user's manufacturing site.

Biocompatibility is the next aspect that should be addressed following system design. To ensure biological safety, products should be manufactured from biologically inert materials and individual components that are in contact with the fluid path should be tested against United States Pharmacopeia (USP) Class VI. Most suppliers will also provide information related to potential extractables and leachables. However, in many cases there will still be a need for further testing with the process fluid under worst case process conditions, especially in instances where there is a long contact time and/or a high surface contact area.

For instance, during the validation of disposable sterilization filtration sets, to accurately represent process conditions, all materials must be gamma irradiated (if that is the chosen mode of sterilization) prior to extractables, adsorption and microbial challenge studies. This is crucial because even though materials such as polyethersulfone (PES), polyvinylidenedifluoride (PVDF), nylon and stabilized polypropylene are known for their low level of extractables, the sterilization method may impact the overall value.

The validation strategy for a disposable membrane chromatography device is much simpler compared with, for example, standard chromatography using gel in a column. The process efficiency will need to be validated as well as leachables. However, neither column packing nor reuse studies must be performed, greatly simplifying this validation step. One rule of thumb users should keep in mind during validation is that leachables testing must be performed for each product and application.

The purpose of the following validation study on a disposable filtration set was to measure and identify the extractables in a process involving the sterile filtration of a solution through a sterilizing grade filter and subsequent storage over a period of time in a bag. Parameters that were examined include:

• nature of the solution

• fill volume (volume versus area ratio)

• storage time

• storage temperature

• potential use of detergent spray.

To achieve this, a scaled-down version of the full size system (Figure 1) was built. One of the objectives was to make sure that tubing lengths for all different storage time points were kept the same. The disposable set was gamma irradiated at a minimum dose of 25 Kgy. The four different aqueous solutions (water for injection [WFI] pH 9.0; WFI pH 2.0; WFI pH 7.0; and 1.0 M NaOH) were tested and extractables were measured using conventional HPLC methods.

Figure 1. Design of the extractables design study for a filter-bag assembly.

The results obtained (Figure 2) showed that whatever the storage time, the extractables were very low. In all cases, extractables were shown to be <120 ppb. Of the four solutions tested, a high proportion of the extractables was found in bag 1, corresponding to the bag that received most of the extractables coming from the filter. This is logical because this filter is the component in the assembly with the highest surface area.

Figure 2. Total extractables from a filter-bag assembly.

With all solutions tested, the level of extractables varied slightly with time. More than 70 chemical compounds were identified during this study. The relative level of each compound depended on the solution that was tested as well as storage time.

This study helped to understand how to design validation studies related to extractables with complex disposable assemblies. A scaled-down system was found to be the best approach, and the four solvents selected in the study were representative of a number of buffer solutions that were evaluated. It also demonstrated that the longest contact time could be chosen as the only test point. Given the extreme sensitivity of the analytical methods used in this test, it is essential to define a reasonable detection limit.

Filtration of a radiolabelled product

Best-suited applications

The availability of disposable technology (Figure 3) on the upstream and downstream side of products from cell culture, and from small-scale to large-scale marks a new era for the manufacture of biopharmaceutical drugs. These technologies serve to speed time-to-market of new products, while enabling continuous improvement of existing processes through retrofitting, which can be done with a minimum of re-engineering and often with little validation because of material consistency.

Figure 3. Typical biotech process - where to use disposables.

On the upstream side, the availability of disposable sterilizing grade filters with mycoplasma removal capabilities has created new opportunities for significant time and cost savings; serum and media manufacturers largely use this technology. A study has compared processing times between a single-use filtration system and a stainless steel assembly using multiple 30 in filter elements.¹ The results showed that the disposable filters not only reduced the operation time by more than half (from 19 h to 8 h), but they also showed that the filtration line could be set up in less than 40 days compared with the 6–8 months it would have taken with a standard approach.

A steady flow of innovative technologies for downstream applications is also being introduced, which includes

• direct flow filtration (DFF) filters from clarification/prefiltration down to virus removal applications

• tangential flow filtration (TFF) cassettes

• membrane chromatography capsules

• filling equipment

• aseptic connections devices

• tubing

• adaptors

• clamps

• bags (up to 2000 L).

Disposable ion exchange membrane chromatography is an ideal alternative to traditional chromatography columns for a number applications. It is particularly effective in large molecule applications because the membrane's large convective pores permit rapid mass transfer. The result is much higher volumetric throughput (up to 100 times faster) and high dynamic binding capacity regardless of molecular weight. Anion exchange membrane chromatography has been successfully applied to the removal of DNA and viruses from a recombinant protein.² The pleated anion exchange membrane is enclosed in a capsule format and is able to effectively reduce the DNA content by 6 Log at a flow rate of 10 membrane volume/min.

Virus particle filtration

As a prepacked single-use device, neither packing validation nor cleaning and reuse studies are required. The robustness of the polishing method eliminates the need to test the DNA content in each batch, contributing to the simplification of the batch release procedure. Both FDA and European Agency for the Evaluation of Medicinal Products have successfully audited the process.

A large number of companies are developing new prophylactic and therapeutic vaccines as well as gene therapies based on viral vectors. Because of the vectors' large molecular size, conventional chromatography supports cannot provide the necessary high dynamic capacity. Disposable membrane chromatography is particularly advantageous in this application because it can achieve high throughput while offering significant convenience to the end user.

Disposable anion exchange chromatography has effectively been used for the purification of an adeno-associated virus (AAV) from a baculovirus/insect cell system.³

In this example, the clarified cell culture containing the AAV was loaded at a flow rate of 10 membrane volume/min on a Q membrane chromatography disposable unit. After initial optimization on a 0.35 mL unit, the process was scaled up to a 10 mL disposable capsule unit. The virus was recovered with a 73% yield while the purity was greatly increased as more than 2 Log of physical baculovirus particles were eliminated as well as a significant proportion of the contaminating proteins and DNA. The whole purification process was conducted in less than an hour.

DFF systems

Demand for integration of disposable filters in increasingly complex assemblies has grown with the availability of a greater variety of single-use filters. For a number of years, biopharmaceutical manufacturers used filters connected to bags for the sterile filtration of cell culture media and buffers. However, strengthening confidence in the technology is extending its use to include filtration of the product itself as shown in the sidebars ('Virus particle filtration' and 'Filtration of a radiolabelled product') (Figure 4).

Figure 4. Small-scale disposable filtration of virus particles.

Conclusion

Disposable systems not only simplify everyday operator tasks, but they also decrease the burden of validation, allowing the biopharmaceutical industry to concentrate on its core activity — developing and producing new drugs that will bring significant health advantages to the world population. The steady rise of new disposable technologies will turn the vision of a completely disposable plastic factory into reality. These technologies will also give small companies direct access to manufacturing capabilities for the first time, while achieving compliance with the most stringent regulatory standards.

Hélène Pora, PhD is marketing director at Pall Life Sciences, France.

References

1. H. Haughney and J. Hutchinson, GEN 24(8), 52–56 (2004).

2. J. Martin, Case study : Othogonal membrane technology for viral and DNA clearance, presentation at PDA International Congress (1–3 March, 2005, Rome, Italy).

3. D. Bataille et al., Evaluation of Mustang (R) ion exchange capsules for the purification of rAAV2/1 produced by the baculovirus/insect cell system, presentation at Baculovirus Insect Cell Culture 2005 (21–24 February, Savanah, GA, USA).