What Are Disposable Technologies

Published Date: 02 Nov 2017

In the past number of years there has been a move towards the use of disposable technology with in the biopharmaceutical industry. There has been research conducted that indicates that over half of biotechnology manufacturers are considering implementing these technologies. (Sandle T 2011). Disposable technologies are designed to replace traditional equipment in all parts of biopharmaceutical production. They are available in many different formats and are generally manufactured from plastic polymers involving processes of injection moulding, extruding and blow moulding. The assemble of these materials is carried out in an ISO 14644 Class 5 clean rooms, these items are then sterilised using gamma irradiation. (Sandle T 2011). The majority of these technologies are being developed to be used in the manufacture of sterile products particularly aseptically filled products. Initially the single use systems that were developed for biotechnological applications were with large-scale bioprocessing for sterile cell culture media and process buffer storage applications. The first disposable technology that was used in large scale production was the WAVE bioreactor in this was in 1996. The first disposable stirred tank reactor was launched in 2005 by Hyclone and this had a working volume of 250L. This system was designed as a self-contained plastic bag that was then placed in a stainless steel shell. (Shukla A 2012) In the last number of years interest in this technology has increased greatly and the use of disposable mixing systems has increased by over 50 per cent in the past 5 years. (Sandle T 2011). These disposable mixing systems have now been designed to hold up to 2000L within a stirred tank bioreactor and there are also various fermenters where mixing is achieved by rocking or orbital shaking. As well as disposable bioreactors there are currently a large number of other technologies that are being developed including tubing, capsules, single use ion exchange membrane chromatography devices, single-use mixers and bioreactors, product holding sterile bags, connection devices and sampling receptacles. (Sandle T 2011) There are 3 main reasons that research and development in to single use technologies is being carried out. These are to reduce processing time which in turn reduces manufacturing costs, to go for manufacturing systems that are more reliable, flexible, cost effective and finally to seek improved sterility assurance. (Sandle T 2011). The implementation of disposable technologies also complements the trend for higher titres of protein as this results in a lower volume of liquid for processing and when using disposable technologies it is easier to change equipment size.

Since 2004 the number of companies that have started using disposable technologies has been increasing, and it is predicted within the next three to five years they will have a 20% share of the biopharmaceutical technologies market. It is estimated that up to 90% of manufacturers and contract research organisation currently use disposable filters and tubing at some stage throughout there process with 77% using disposable filters and with 58% using disposable membrane absorbers. (Shukla A 2012).

3.0 Advantages of Disposable Technologies

There are a number of advantages to implementing single use technologies throughout biopharmaceutical production, these include the elimination of the need for cleaning and sterilisation of the equipment which in turn reduces the use of cleaning chemicals and water for injection, a reduction in the storage requirements needed, a lowering of the process time needed for manufacture, an increase in the process flexibility and also a reduction in the risk of microbial and cross contamination.

3.1 Cleaning and Sterilisation

Cleaning and sanitising of equipment has long been an expensive and time consuming part of biopharmaceutical production and there are a number of regulatory requirements that companies have to adhere to with regard to this step. The main objective of a cleaning and sanitisation step is to ensure there is no contamination or denaturalization of the drug product as a result the use of contaminated equipment. The manufacturer needs to develop their cleaning and sanitisation step taking into account the products that they are producing to ensure that there process is adequately removing all traces of the product and any other contaminants that may be present. This step also needs to be validated taking into account the variety of products that are being manufactured and also the frequency the cleaning step is performed, is it between each batch or just when changing from one product to another. This is carried out to ensure that there is no chance of cross contamination between batches and also that the cleaning products are being effectively rinsed away before the equipment is used. There is also a cost factor when using a cleaning and sanitisation step, as the equipment is cleaned there is a large amount of water for injection, steam for injection and assorted cleaning products used which add an extra cost to the unit price of the batch. This step also results in a large amount of down time and manpower as the equipment is dismantled, cleaned, sterilised and then reassembled aseptically.

The removal of this step is one of the major advantages of using disposable technologies. This reduces the amount of water for injection and cleaning products used in the manufacturing and it also reduces the amount of time that would be traditionally needed to set up for another batch. The removal of the cleaning validation step from day to day processing has been estimated to reduce the water for injection needed by 80% and also give a saving in electricity of approximately 72% (Pigeon T). The removal of this cleaning step also reduces the processing time as there is less downtime between batches. Another report estimates that there is 87% less water, 21% less labour 38% less space and 29% less energy needed in using disposable technologies in comparison to there more conventional counterparts.( Valle C). A study carried out comparing a traditional stainless steel system designed to produce a typical monoclonal antibody at a scale of up to 1000 litres compared to a disposable system designed to accommodate the same product showed that the cleaning step of the system was responsible for using an extra 4929 MJ of energy per campaign in addition to this there was an extra 1968 MJ of energy used in the sterilisation step. (Rawlings B 2009) Another benefit with the removal in the cleaning and sanitisation step is that is also allows for the removal of its associated validation and the development of its methods. It is estimated that this can reduce the development time of a product by up to 2 months (Pigeon T).

To date there have been no FDA guidelines with regard to the used of disposable technologies within biopharmaceutical process, however the International Conference on Harmonization ( ICH) Q7A document, Good Guidance for Active Pharmaceutical Ingredients sections 5B 6B and 12 specifies the requirements for equipment maintenance and cleaning, documentation of the procedure and also validation of the procedures. This document takes into consideration the use of disposable technologies and advises that when they are used the need for cleaning validation and its associated documentation are not required. (Cleaves K 2003)

3.2 Storage Requirements

3.3 Increased Process Flexibility

3.4 Cross Contamination

Traditional technologies can be a source of cross contamination. This can be caused by the equipment not being cleaned properly and the drug substance being contaminated by the preceding product, as disposable technologies are only used once there use eliminates any chance of this cross contamination.

3.5 Health and Safety

Some biopharmaceutical production involves the use of cytotoxic drugs or other hazardous material that is harmful to the operators involved in production. Disposable technologies offer a large amount of protection for these operators as the systems can be designed to be closed there is no point in the process where the operators have to interact with the product and also as the system is discarded after use there is no dismantling of the system for cleaning as would be seen with traditional systems.

Disposable systems can also be designed to be more ergonomic to use than traditional systems and as they are smaller they are easier to move and set up. Millipore have designed there systems so be as user friendly as possible and have even designed the tubing size to be just the correct length for an operator that is wearing double gloves to use, if this tubing was too long then it would cause problems when it was being cut and if it was too short it wouls be too difficult to handle. They have also designed there systems to be as space efficient as possible and there Mobius Flex Ready Solutions have interchangeable filters that can be used with all of the systems, they are also designed so that one cart will work will all of the systems. To make the systems even more user friendly they process container carrier is tiltable to 90 degrees therefore making it more ergonomic for people of all heights to use it and the feed pump sits at an angle to allow the process container to be drained completely thereby removing the chance of any expensive protein or cytotoxic chemical being left behind. ( Valle C)

3.6 Cost Saving

Biopharmaceuticals are expensive to produce; when they were first developed this cost was largely ignored as the price that the product could be sold for more than made up for the high product manufacturing costs, in some cases the profit margins exceeded 98% ( Shukla A 2012). In recent years there has been a slowing down of biopharmaceutical drug discovery and this coupled with an increased number of manufacturers producing biopharmaceuticals and an increased development of biosimilars has driven the price of biopharmaceuticals down and so manufacturers have to reduce their production price to make a profit. Currently a contract manufacturer makes a 33% profit when manufacturing a biopharmaceutical and a biopharmaceutical company that contains an inbuilt manufacturing facility can spend up to 25% of its operational costs on manufacturing costs. (Charles I 2007)

There are a number of different ways that disposable technologies can be cited as cost saving. To set up than traditional technologies is expensive as the equipment itself is costly to buy and needs time to be set up, the disposable technology is cheaper to buy and much quicker to install. It costa $10 million to build, kit out and launch a pilot plant that is designed for volumes up to 100 litres, this cost is $40 million for a 1000 litre plant and for a plant designed to accommodate volumes up to 20,000 litres this cost may be as high as several million dollars. (Charles I 2001). It has been estimated that designing a new facility using disposable technology instead of traditional technology can reduce the capital cost by up to 40% (Shukla A 2012). This cost difference is even greater when scaling up a product as with each step up will require larger piece of equipment to be used resulting in a more costly and lengthy changeover when using traditional rather than disposable technologies. Once the facility is up and running there can also be cost saving seen as the use of disposable technologies allows a faster turnaround time between campaigns as there is no need for a cleaning and sanitisation step and a faster production time as it reduces bottlenecks throughout the process, these cost saving however need to be balanced against the extra costs incurred by the purchasing of new equipment for each campaign. Disposable technologies have also been seen to reduce the energy requirements needed for a facility this is mainly due to the removal of a cleaning and sterilisation step as the production of water for injection and purified steam is very energy consuming. A study carried out comparing the energy consumption using a typical stainless steel system, such as one used for monoclonal production processes at the 1000 litre scale, with a disposable system designed to accommodate the same product and volume. This system had flexible bio containers to replace the stainless steel buffer and storage tanks, the vent filters used in the stainless steel model were removed, the liquid filters were replaced with disposable capsule filters, and the exhaust-gas filters were replaced by disposable capsule filters. In the downstream process the capture and ion exchange systems used for polishing were replaced with disposable membrane adsorber capsules. When assessing the energy requirement there were three functions used these were cost of sterilisation, cost of cleaning and the cost of disposing of waste equipment and also the containers it was delivered in. Sterilisation was carried out by purified steam for the traditional system and by gamma irradiation for the components of the disposable system. Cleaning was performed using agents such as 1M sodium Hydroxide and 1M phosphoric acid and used water for injection for the stainless steel system the disposable system did not need any cleaning step. With the materials calculation the waste raw material per batch was calculated by dividing the equipment weight by the number of runs that it can be used for this for the stainless steel for the disposable system it is discarded after each use so the weight of the equipment was the weight of the waste. Other functions that may also produce energy consumption such as transport and installation costs were not included in this study. When the study was assessed it determined that the energy requirements for the disposable system was about half that seen with the traditional system. The values were 8018 MJ for the traditional system and 4156 MJ for the disposable system. (Rawlings B 2009) Energy levels can be further reduced when the manufacturer could elect those certain procedures such as disposable connection to be performed within environment that do not need a unidirectional airflow cabinet. (Sandle T 2011)

It has also been noted that depending on the product value there may be a cost saving seen from the elimination of contamination leading to batches being rejected and process downtime. (Sandle T 2011)

3.7 Process Analytical Technology (PAT)

3.8 Increased Sterility Assurance

When producing a drug product the process must be designed in such a way that the finished product is free from bacteria, viruses or other potentially adventitious agents. This is usually carried out by designing a functioning cleaning and sanitisation step and in some cases a final sterilisation step by autoclaving. As biopharmaceutical products are protein based they are heat labile and this means that the they cannot be a final heat sterilisation step in the process. This increased the reliance that manufacturers have on their cleaning and sanitising step until the advent of disposable technology. As disposable technologies are bought in sterile and disposed of after each use their use reduces the possibility of cross transference of microorganism while also minimise the risk of microbial contamination from the environment and also removes the risk of the process becoming contaminated due to a failed or invalid cleaning and sanitisation step. Disposable technologies are usually sterilised using gamma irradiation; this gamma irradiation kills bacteria by breaking down bacterial DNA and inhibiting bacterial division. This also safeguards the plastic used in the manufacture of disposable technologies from degradation. Sterilisation cycles used in disposable technologies are designed to achieve a Sterility Assurance Level 10-6. (Sandle T 2011) This sterility assurance is increased by the way that the connections are undertaken and often with the number of connections that are required to be performed. (Sandle T 2011)

3.9 Process Efficiencies

The use of single use technologies can increase the process efficiencies in a number of ways. It can reduce bottlenecks that normally seen when using traditional technology in a process; these bottlenecks are usually caused by long turnaround times due to cleaning and sanitisation of equipment and vessel capacity. The removal of a cleaning and sanitisation step when using disposable technologies also allows for a shorter down time between campaign and reduces the need for cleaning reagents, water for injection and purified steam that are traditionally used in the cleaning step for stainless steel equipment. This also leads to a decrease in the amount of energy used. As disposable technologies are cheaper to set up and do not need a large amount of space for storage when not in use a number of different vessels, of the same of varying sizes, can be stored on site for use in throughout the process allow a quicker turnaround time. Disposable technologies can also be more easily adapted to suit the process as companies are constantly developing new systems designed to synergise with the existing biomedical layouts of different production operations. Disposable systems can also be delivered preassembled reducing the work involved in the setup of the equipment and reducing the amount of errors that may occur. When disposable technologies are aligned with QbD approaches it can be used to streamline processes and when new processes are being developed less large scale capital equipment needs to be sourced. The use of disposable technologies also simplifies the changeover of equipment from one process to another

4.0 Disadvantages of Single Use Technologies

There is a number of different validation steps that need to be considered before single use technologies these include assessing any leachables or extractables that might arise when the product comes into contact with the single-use technology. The availability of the technology also need to be assessed and the development costs. (Sandle T 2011)

4.1 Validation

When a manufacturer decides on implementing disposable technology in there manufacturing process there are number of different validation test that should be per formed to ensure that the technology that they are implementing is fit for its purpose. One of the first things that they should assess is whether or not the equipment is compatible for use with the chemicals that it will be holding. There are very few standard tests outlined for this assessment but an adaptation of ASTM D543-06 "Standard Practices for Evaluation of the Resistance of Plastics to Chemicals" can be used in grading the compatibility of the equipment. The material should be assessed, before and after it has been exposed to the solvent of use, for changes in weight, dimensions, colour, surface quality, appearance and strength to determine if the polymer is compatible with the solvent. Functional tests should be carried out to ensure that the product is capable of withstanding normal day to day use, this test will vary depending on the piece of equipment for example an integrity test could be carried out on a bioreactor. In this type of test an action that would be defined as normal such as the opening or closing of a connector should be performed to ensure that it does not affect the integrity of the bioreactor. (Eibi R 2011)

If the equipment needs to be sterile before used then the sterility of the equipment should also be assessed before it can be deemed acceptable for use. Sterilisation of disposable technologies is carried out by gamma radiation and the minimum dose that is necessary to ensure that there is complete sterilisation at 10-6 sterility assurance level should be validated. International Organisation for Standardisation (ISO) 11137 standard outlines methods for carrying out this validation. (Eibi R 2011) There are also a number of different contaminants that may be present in disposable equipment including particulates and endotoxins and the level of contaminants that is acceptable depends on the purpose of the equipment. If the equipment is to be used before a filtration or purification step then the requirement are less stringent as the contaminants are likely to be removed during this step. The amount of particulates present should be low enough to give a product that is compliant with the United Stated Pharmacopeia (USP) 788. For equipment such as bags and cell culture vessels this can be tested by addition of the minimum volume that can be used in the bag and checking this for compliance. Testing for endotoxins is carried out by USP method no 85 and when carrying out this test it is important that the complete assembly. (Eibi R 2011) There should also be an expiry date given to the equipment as polymeric materials are known to age and over time there physical and compatibility characteristics may change. ASTM F1980 outlines how accelerated studies can be carried out. When carrying out these tests on sterile products they should be undertaken after gamma radiation as this can alter the properties of the polymeric materials. Long term studies should be carried out as they can provide information on tests that may not be feasible with the accelerated studies such as sterility assurance and these studies can also be used as conformation of the results seen with the accelerated studies. (Eibi R 2011)

Along with these validation tests there are a number of other tests that are performed routinely during incoming quality inspection such as integrity tests, sealing strength and a visual check and dimension. (Eibi R 2011)

Bioprocess containers are tested according to USP Class 6 standards before use and the vendor confirms the validation and performs the following tests when the containers are purchased, acute stem toxicity, intracutaneous toxicity, implantation test, cytotoxicity (agar diffusion and elution) endotoxin level, heavy metals concentration, buffering capacity, non-volatile residue and residue on ignition. (Charles I 2007).

4.2 Leachables and Extractables

In a review of contract manufacturer and biomanufacturers it was noted that the highest risk they saw with the move to disposable technologies was the risk of leachables contaminating there drug products. This risk is further compounded by the fact that disposable technologies are a new development and as such manufactures are inexperienced with the components used to make up the equipment and how is can react with the solvents used throughout the manufacturing process and also with the drug product itself. There is also a regulatory requirement to ensure that leachables do not affect the drug quality, as outlined in the US FDA’s GMP requirements for finished pharmaceuticals states that "equipment shall be constructed so that surfaces that contact components, in process materials or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements" (Ding W 2008). Regulatory approvals require that the process itself be validated to ensure there are no adverse effects on the product by the use of the disposable technology. This involves samples taken at different stages throughout the process being tested for plastic leachables and extractables along with toxicity studies of the plastics involved including implantation in laboratory animals. This full validation may cost over $100,000 dollars for each major contact plastic that is tested. There may also be chemical analysis that can identify many polymer leachates which may already have a number of toxicology studies and safety assessment carried out on them. (Rader R 2012)

Leachables can be defined as substances that will diffuse out under normal routine conditions they can be described as migrants from the component material and can often but not always be a subset of extractables. Extractables are substances that will leach out under extreme experimental conditions they can be produced in organic, aqueous or dried product vehicles they are useful as they can help with predicting the likely leachables that may occur. There are a number of different ways that leachables and extractables can interact with the drug product however they frequently have well defined breakdown patterns that can be characteristic of the material degrading, this is useful when assessing new materials. Extractables and leachables are most commonly associated with polymeric and elastomeric materials and the greater use of additives with these compounds to increase their stability and to aid in the formation of the material components increases the amount and type of leachables that can be released. Compounds such as polyethylene may have up to three antioxidants or stabilizers along with at least one processing aid usually lubricants or antislip additives. ( Bestwick D ). For all new biopharmaceutical products a comprehensive assessment must be carried out for biological licence submissions (BLA) this needs to include an analysis of the overall production process for the products, and a plan for data collection from early clinical trials material production through to the BLA submission and right through until new drug applications. Leaching can occur in a number of areas throughout the biopharmaceutical processing, in upstream processes including media and buffer preparation and also the cell culture or fermentations step. In the downstream processes it can occur in the concentration steps or throughout the exchange and purification operations. The storage steps also need to be assessed as there is a possibility of leaching during bulk storage operations and also in the drug product storages such as the vials and pre filled syringes. Most suppliers have comprehensive data on the extractables released from there equipment usually using a common solvent such as alcohol or water. This data usually contains the profile of any extractables present and also the individual chemical species. The type of leachables that may be present can be determined by accessing these studies and this can be then implemented into the manufacturers own test protocols and also into regulatory submissions (Smart N 2012)

When determining an acceptable level for leachables in a drug product there are a number of different factors that need to be considered. Some things include in this assessment would be the route of administration and also whether the drug is for acute conditions or chronic conditions. This purpose of the drug is an important factor as patients with chronic conditions will be taking the drug more regularly and so will be accumulating more of the contaminants over time. ( Smart N )

The biopharmaceutical industry has already had problems with leachables, Ortho Biologics manufacture an EPO product called Eprex recombination erythropoietin that caused more than 300 deaths in Europe. The product was reformulated and it is believed that one or more substances that leached from the rubber used in the prefilled syringes then altered the structure of the reformulated product. This altered structure caused patients to develop antibodies to EPO, both to their own and also to the injected substance. ( Rader R 2012)There was also a study published that outlined how a parenteral drug that switched from being manufactured to contain human serum albumin to polysorbate 80 caused a new contaminant to be leached from the final container. This leachable then resulted in a high incidence of antibody positive pure cell aplasia in patients. (Bestwick D)

As well as patient safety leachables can impact product stability and efficacy and this must be assessed by the manufacturer.

4.2 Compatibility with the product

Before disposable technologies can be implemented in to the process they must be tested to ensure that they are compatible with the product. A chemical reaction could cause the product to react with the plastic causing contaminates to be released from the polymers and the possibility of these occurring increases with the length of time that the product is in contact with the plastic. The types, causes and effects of these contaminates is outlined in section The optimal conditions would be that the plastic would be chemically inert and at present there are several manufacturers that are developing plastic materials that are designed so as not to react with a large range of different chemicals and formulations. The physiochemical properties are examined based on assessing a number of chemical and physical properties usually based on USP Physiochemical Testing (<661> Containers-physiochemical tests-plastics) guidelines. The EP also outlines specific tests that are applicable to silicone (Ph Eur 3.1.9). The assessments used depend on the plastic material and also on the product that the plastic comes into contact with and they involve running a formal study, it is usually recommended that this study is run in conjunction with the vendor of the equipment as the vendor will have data that relates to the chemical composition of the material. Phytochemical testing is carried out by measuring the measuring the properties of impurities extracted from plastics when they are leached into a suitable extraction medium, isopropyl alcohol or purified water can be used as the extraction medium. The equipment should be tested after it is gamma radiated as this step can cause a significant amount of deterioration to the materials. The testing is carried out over a specific length of time and at a set temperature. These are usually based on the storage time of the product and the storage conditions at which it is held. The extraction media must then be tested for contaminants such as heavy metals, buffer capacity and non-volatile residues, as well as other changes that may have occurred in the equipment such as a change in colour or a loss of material strength. The manufacturer should also assess whether the disposable technology could be damaged by the manufacturing process such as pressure, mechanical force and temperature. (Sandle T 2011)

4.4 Safety

One of the primary concerns of a biopharmaceutical company is that its product is safe to use. Traditional equipment is manufactured using stainless steel and this material is designed to be inert and not to react with the solution that it may be holding or transferring, this material has also been in use for sufficient time for manufacturers to have legacy information on its safety information. Disposable technologies are manufactured using a mixture of plastic polymers, and to date there is not a large amount of information gathered on their safety information.

Many of the polymers that are being used in these technologies are polymers that have been used in medical devices or pharmaceutical packaging for many decades and are generally recognised as safe, however the standards and test methods that were used to approve these methods may not be as up to date as the current standards and methods and so these the standards used for testing these products may be inappropriate for use that involves product contact. There is also a lack of modern studies and assessments that may be required by regulatory bodies to allow use in biopharmaceutical manufacturing for example there are only a few polymers that would contain the full spectrum of chemistry and toxicology date such as multiyear carcinogenicity and reproduction studies which would be needed to full assess the impact of using them as a product contact equipment. There is also a lack assessments carried out on the polymer leachates and the by-products that may occur during gamma radiation. Finally there is a lack of information on long term patient exposure. FDA regulations towards the use of disposable technologies had so far been reasonable and they have not applied the stricter standard that is usually seen when using polymers in areas such as medical devices and food packaging. However this standard could be raised in the future if there are toxicity problems seen when using disposable technologies. As well as with the use of these known polymers manufacturers also have to take into account that there may be a large number of unknown plastics in there equipment. Manufacturers do not always list components such as tie layers or adhesives that may be used to construct the multilayer laminated bags and liners and these may be a cause of leachables in the product. To date there has only been a minority of suppliers that have documented there plastics and parts supply chain to allow the manufacturers to determine what is actually in there equipment. ( Rader R 2012)

4.5 Availability of Technology

As disposable technology is a relatively new development there will obviously be issues with the availability of the technology One of the problems with the use of disposable technology is scaling up especially with the use of bioreactors For example a GE Wave bag has a maximum scale of 500 litres, if you need a higher volume size than this you then need to invest in more hardware such as bag holders, internal mixers which makes the systems more complex and less disposable. (Netterwald J). However it should be noted that larger bag sizes have been reported including a 10.000 Litre bag that has been marketed by HyClone, and that there is also the option of using disposable bags as inner liners for less expensive solid wall vessels that do not require electro polished 316 stainless steel (Fox S) allowing for a cheaper initial set up and also removing the need for a cleaning and sanitisation step.

Another limitation with the use of disposable technologies is the lack of supplier options for example there are only a handful of suppliers that offer disposable bioreactors and only two that offer disposable centrifuges. (Shukla A 2012)

Disposable technologies may not be suitable for all biopharmaceutical processes at the moment single use microbial fermenters have been limited to a scale of 50 litres as at a larger size they are unable to provide adequate mass transfer needed for complete growth and protein production and they are unable to deal with the heat that is produced during fermentation.

4.6 Supplier Issues

Another limitation with the use of disposable technologies is the lack of supplier options for example there are only a handful of suppliers that offer disposable bioreactors and only two that offer disposable centrifuges. (Shukla A 2012)

4.6 Limitations of the technology

With regard to the limitation of the technology it has been noted that disposable bioreactors are not practical when using E coli or some yeasts. This is due to the high biomass levels that are seen during fermentation which cause mixing and oxygenation problems. (Netterwald J) Also not all cell lines are compatible with the use of this technology, there have been studies carried out using and antibody producing NSO cell line that did not grow in Wave bioreactors in the presence of chemically defined serum-free growth media that contained cholesterol which was added to the serum as a supplement. The cells were found to grow poorly and adhere to the surface of the bag, it is presumed that this was caused by the cholesterol that was present in the serum as when cells were used that did not require the presence of cholesterol this problem was resolved. (Charles I 2007)

There are also limitations with the use of disposable centrifuges for harvesting the product after fermentation. ( Netterwald J)

4.7 Standardisation of Materials used

Each supplier that manufactures disposable technology have their own materials and grade of materials that they use and so all disposable technology may not be created equal.

4.8 Environmental Impact

In the past number of year’s biopharmaceutical producers have placed a much larger emphasis on environment issues caused by factors such as worries about environmental change, limits being places on waste emissions, thee rising costs of waste disposal and the greater restrictions being placed on waste disposal. The environmental impact of a move to a disposable set up needs to be assessed when a company is thinking of installing single use systems. There are a number of different factors that need to be considered when assessing the overall environmental impact. Both the amount and the quality of the waste needs to be assessed. As well as the equipment there is also the associated packaging waste. It has been reported that there is approximately 880kg of solid waste generated for each batch when running a completely disposable process, both upstream and downstream when manufacturing a 3x2000L scale batch. ( Eibi R 2011) Packaging waste will not be contaminated by the product and so can be recycled if possible or sent to landfill. The equipment however needs to be dealt with separately. As disposable technologies are used once and then disposed of, there disposal method needs to be assessed to ensure that it is suitable for the chemicals that were used in the equipment. There are a number of different The production of this extra waste can however be negated by the removal of the cleaning and validation step that would be needed if traditional technologies were implemented, as this the energy needed for the production of water for injection and purified steam along with the used of harsh chemicals would also impact the environment.

Manufacturers need to assess the environmental impact causes by both of this step to determine which is more sustainable for their facility.

5.0 Uses of Disposable Technologies

Disposable technologies are being developed to replace traditional equipment at all stages throughout the biopharmaceutical process from cell culture to final fill.

5.1 Aseptic Connections

An aseptic connection can allow liquid to be transferred from one container to another, such as the addition of media to a bioreactor, without risk of microbial contamination. Traditionally connections between equipment were carried out by welding the pipes together or clamping. This step was a potential contamination problem as there was a risk of microbial contamination from the environment or from the operator. Disposable aseptic connections remove these risks. There are a number of different systems that manufactures can choose between if they are looking for aseptic connections. The first thing that must be established is whether an open or close connection is needed. Open systems include Luer Lok manufactured by Value Plastics, sanitary tri clamps or quick connects whereas closed systems used where an aseptic connection is needed include Kleenpak sterile connectors manufactured by pall or ReadyMate DAC manufactured by GE Healthcare. (Charles I 2007) According to Pall there Kleenpak connectors are quicker to assemble, are shape coded to ensure that the connection is properly connected, and they also contain a ratchet mechanism that prevents the connector from disassembly.(Cleaves K 2003) The tubes are designed so that two non-sterile ends are pressed together, this moves the ends and creates a sterile flow path, these connections cannot be disconnected without breaking the sterility of the flow path. ( Eibi R 2011) Another option for sterile connections is tube welding and these tube welders can be sourced from companies such as GE Healthcare, Satorius and Terumo. (Shukla A 2012). With this procedure a hot blade cuts through two silicone hoses and the heated ends are pressed together to allow them to bond, this system has an added advantage in that it can be performed in non-sterile surroundings because of this most disposable manufacturers has realised the benefit of these connections and have produced bags that have these thermoplastic silicone hoses. (Eibi R 2011) Aseptic connections have been developed to allow for a totally enclosed and automated process that allows a connection to be performed outside a unidirectional airflow cabinet while remaining sterile. These systems are also designed to allow a fast and safe disconnection that removes any chance of backflow occurring. (Sandle T 2011). Before a manufacturer implements disposable aseptic connectors they must be assessed through a bacterial challenge test designed to determine if bacteria can breach the connector seal and thereby contaminate the liquid being transferred. There are a number of ways in which this test can be carried out. Firstly a suitable strain of bacteria must be selected, this bacteria must be smaller than average to ensure a worst case scenario is being assessed. This bacterium would be cultured to produce a high concentration broth culture and a sterile connector would be immersed in it for up to 5 minutes. This connector would then be used to transfer sterile media into a sterile container, which would then be incubated for 7 days to allow any bacteria that may have transferred through the connector to grow. (Sandle T 2011)

Aseptic connections are not only used for connecting tubes they are also useful as aseptic ports for disposable bioreactors and other such systems. Thermo Fisher Scientific have coupled the Pall Kleenpak Sterile Connectors with the Hyclone system, this allows conventional pH meter and oxygen probes to be used in the disposable bioreactor.

In some cases there is also a need for the system to be disconnected aseptically. There are a number of ways that this can be carried out. If the system was originally connected using quick connect fitting these can be easily disconnected under laminar flow condition. The open ends are then reclosed with aseptic end caps and it is then recommended that there is a disinfection process carried out. This system theoretically allows the systems to be divided or disconnected aseptically between two clamps or bio valves but in practice there is a high risk of infection caused by leaking clamps or operator error. Are more widely used method of asepticl disconnection is by using sealing ir crimping methods, With this method the thermoplastic tubes are sealed by using heat and pressure to melt them and force them together. The temperature needed to fuse the tubes id generated using an electrical or radio frequency. This procedure can be carried out in any environment. With the crimping method a crimping tool is used to cut the sleeve and the tube after crimping leaving both ends of the cut tube securely closed nu the two halves of the sleeve. There is also another very simole method of carrying out an aseptic disconnection the line is secured with two cable ties and a cut is made between them.( Eibl R 2011)

5.2 Holding Systems

Throughout the biopharmaceutical production process there are a number of different steps that involve the storage or transportation of liquids or powders this can be the product itself or reagents used in the process such as buffer or media components. Traditional holding systems were fixed stainless steel containers that were cumbersome to move, complicated and required a lot of extra separate valves and piping. Disposable holding systems are usually plastic bags or packs, these take up less space in storage and do not require a cleaning and sterilisation step and so reduce processing time or the product. Stainless steel vessels have been reported to take 8-10 hours in their cleaning and sterilisation step whereas when disposable systems are used this is removed. (Sandle T 2011) Disposable product handling systems are usually designed to consist of two or three dimensional bags that are connected to a manifold of tubing, connectors and filters. They are designed to ensure that no part of the product will come into contact with any equipment part that is not disposable or does not maintain the pathway of the closed system assembly. (Sandle T 2011) Disposable bags can be used to line container and transport systems in open systems and so do not need to be gamma sterilised. These tank liners are useful when preparing solutions such as media or buffers that are then sterile filtered as they can be easily used with many commercially available overhead mixers. (Eibi R 2011) Bag systems for use in the freezing and thawing of liquid have also been developed. The polymers used in the bags has been shows to be safe to use at low temperatures and manifold filling systems have been developed to allow the liquid to be portioned off before it is filled this allows the user to thaw the required quantity when needed while allowing the remaining liquid to stay frozen. A problem that has been seen with the use of this technology for freeze thawing is the bag integrity. The bag may be damaged while containing the frozen solution and this would not be noticed until the thawing stage. (Eibi R 2011)

There are many different suppliers of bioprocess containers for solution storage or preparation. These containers are usually manufacturers from materials such as polyethylene or ethylene vinyl alcohol and can be sourced in volumes from 25ml to 500 litres. Before use these products should be assessed using microbial inhibition studies using the maximum holding times that will be used for the product to ensure they do not allow the product to become contaminated during use. (Sandle T 2011)

5.3 Sampling Bags and Systems

When manufacturing a sterile liquid there is a regulatory requirement that a sample is taken for bioburden testing from the bulk product before the final filtration step. Traditionally the equipment used in this step was a sterile syringe that was placed into the bulk product to aseptically remove a sample of the liquid and transfer it to a sterile sampling container. This process however is highly operator dependent and involves a large number of steps each of which could be a source of contamination which could then lead to a false positive result being returned. Disposable sampling bags are designed to allow a sample to be taken from the bulk product without contamination and thereby eliminating the chances of a false positive result. Before a manufacturer can implement these biocontainer bags they must ensure that they are compatible with the material the bag is manufactured from as if there are any materials that can be transferred from the bag into the solution that could inhibit microbial growth this will result in a false negative result. This can be assessed by performing a microbial challenge study where the product held in the bag is contaminated with a low level of microorganism and held for a period of time similar to the time taken for the filtration process and then the level of microorganism present is assessed. The absence of inhibition is confirmed by the recovery of the contaminating microorganism at a lever close to the initial level. (Sandle T 2011)

5.4 Tubing

Tubing is used extensively throughout biopharmaceutical process, media needs to be transferred to the bioreactor or fermentation vessel, the cell culture broth needs to be transferred after fermentation to a harvesting step, this then needs to be transferred to downstream processing where extra buffers may need to be added, and finally the drug substance needs to be transferred into its final packaging. Traditionally this movement took place through a series of fixed and detachable connections with valves and fittings made up on stainless steel with polymer gaskets that have been produced under hygiene design conditions. When designing these systems there are a number of different that needs to be considered. There pipes and adaptors should be as short as possible to allow for efficient cleaning and sterilising and to ensure that there is complete self-draining. There should be as little as possible kinks in the systems including narrow gaps, dead legs transitions, and junctions or built in components as these all pose a contamination risk. All parts of the tubing that are to come in contact with the product should be sterilised and this can be carried out using purified steam or other gases such as ethylene oxide, formaldehyde or hydrogen peroxide as a primary step. This sterilisation is a prerequisite for aseptic connections, disconnections, and sampling. During the steam sterilisation step the pipes and other systems are filled with clean steam with the condensate them been conducted away in steam traps. (Eibi R 2011)

The use of disposable tubing removes the need for this sterilisation step, disposable tubing can be used instead of fixed piping for the transfer of all solid liquid of gaseous substances.

5.5 Chromatography and Adsorptive Separators

Traditionally chromatography has been carried out using large stainless steel column that contain a resin that can be removed for cleaning and regeneration. These resins are expensive and so regenerating and reusing them was required to ensure the process was efficient.

Disposable chromatography columns have been developed by companies such as GE, Repligen, Life Technologies, Biorad and BIA Separations and can be purchased pre packed and pre validated thereby eliminating the need for packing and testing of the columns before use.

A disposable column chromatography system on the market developed by GE Healthcare is the Akta Ready. This is a system developed that couples a mixing and storage tank containing a plastic liner inside a stainless steel tank with a chromatography skid developed by GE Healthcare. This system has a completely disposable fluid flow path and so no buffers or product will come into contact with any equipment parts that might require cleaning.

Large columns are still seen as the most useful technology for bind and elute processes but for flow through chromatography steps more and more companies are moving towards disposable membrane cassettes. Membrane chromatography so far has the lowest take up of all the disposable technologies developed at just 19%, however it is the newest technology developed and has the strongest market growth with a compound annual growth of nearly 27% between the years of 2006 and 2012 ( Shukla A 2012)

5.6 Bioreactors

Traditional bioreactors are stainless steel tanks that had to be installed and commissioned before use and cleaned and sterilised between each used. This step increased the turnaround time between each campaign. Disposable bioreactors are plastic bags that can be used on their own or placed into a stainless steel shell and disposed of after each use eliminating the cleaning and sanitisation step and reducing the downtime between campaigns. As they are pre sterilised when purchased they reduce the chance of microbial contamination and as there is a new bag used for each batch they eliminate the chance of cross contamination by another product. In the last number of years interest in this technology has increased greatly and the use of disposable mixing systems has increased by over 50 per cent in the past 5 years. (Sandle T 2011). The use of these disposable bioreactors greatly reduces the amount of time needed between changeover of batches normally it takes between 1 to 4 days turnaround but using disposable technology this can be reduced to a couple of hours. ( Valle C) These disposable mixing systems are now designed to hold up to 2000L within a stirred tank bioreactor and there are also various fermenters where mixing is achieved by rocking or orbital shaking. ( Sandle T 2011)

The first disposable technology that was used in large scale production was the WAVE bioreactor in 1996. This reactor is partially filled with medium and then placed on a tray, this tray can then be rocked to allow for mixing of the contents and to allow adequate gas transfer. These bioreactors can now be purchased in volumes up to 500 litres and are useful in steps such as the initial cell culture inoculum and in scaling up steps. These bags have been proven to be compatible with most cell lines commonly used in biopharmaceutical production. Bioreactors that are orbitally shaken have also been developed, these work on the rotating motion of the culture vessel around a central shaft which allows for mixing of the contents and adequate gas transfer through the media. This type of bioreactor has been shown to have a very high gas transfer rate and so can be used without any oxygen probes; this is because the oxygen supply occurs by air alone. These bioreactors are now available in volumes of up to 200 litres. (Shukla A 2012). Pneumatically mixed bioreactors have also been developed and these are designed to mimic traditional bioreactors where mixing and oxygen are supplied via air bubbles in the system these systems are available from 3 litres to 500 litres. The most common type of disposable bioreactor that has been developed for the production of biopharmaceuticals is the stirred tank bioreactor. This system is designed as a self-contained plastic bag that was then placed in a stainless steel shell. The first of these was launched in 2005 by Hyclone, this system had a working volume of 250L and used a top driven impeller for mixing and agitation. There are a number of different suppliers that now produce disposable bioreactors based on this principal including Xcellerate which manufacture a bioreactor called the XDR, this has a different mixing system to the Hyclone as it has a magnetically coupled bottom driven agitator. These stirred tank bioreactors can typically be used with volumes up to 2000 litres (Shukla A 2012). There are also disposable reactors that use hollow fibre cartridges, with this system the cells grow inside the cartridges around a semi permeable 10 KD-hollow fibre membrane, the semi permeability of this membrane allows for the cells to be fed across it and for waste to be removed. These bioreactors also have the advantage of concentrating the product that is produced by the cells. (Charles I 2007)

Data obtained from a contract manufacturing organisation shows that when wave disposable bags used in conjunction with disposable cell bags wave bioreactor systems cell growth for most cell lines including CHO cells and BHK cells is comparable to that obtained from traditional standalone systems, There was also a similar growth trend observed when the volume was scaled from 20 litres to 200. (Charles I 2007)

5.8 Filtration Systems

5.9 Disposable Sensor Technology

Throughout biopharmaceutical manufacture there are a number of different parameters that need to be monitored to ensure that the process is performing correctly. Some of these parameters include dissolved oxygen present, pH measurement, and total cell concentration in the cell culture or fermentation stage, and pressure sensors for filtration or chromatography stages. Traditionally these parameters we measured using re-useable equipment such as pH meters or DO2 meters that were cleaned and sterilised between each batch and placed directly into the media for constant monitoring of the system. For parameters that do not need to be constantly monitored the samples can be removed from the system and being placed into a measurement system such as a uv spectrophotometer for analysing the protein concentration. When samples are being taken from the system for analysis there is no requirement for sterility of the measurement system as they sample are discarded after use and so there is no advantage of this being replaced by a disposable system, the sampling system however needs to be sterile as this is placed directly into the product stream. Disposable sampling systems are outlined in section

Disposable technology is being developed to replace the traditional systems such as pH meters and DO2 meters that are placed into the product stream as this would eliminate the cleaning and sterilisation step and reduce the chance of microbial contamination of the product The probes used for these sensors are expensive an so it would not be economically viable to replace them after each use.. Disposable sensors have a number of requirements, they need to be autoclavable as they are going to be in contact with the product, inexpensive as they are going to be replaced after each use and usually there is two sensors used at each stage to ensure the reading is correct and reliable. Disposable sensors have been developed that involve the use of an inexpensive disposable probe placed inside the bioreactor or other piece of equipment that is then attached to a traditional piece of analytical equipment outside. A similar system is to use inexpensive disposable sensors that are based on semiconductor devices and placed into the product stream. ( Eibi R 2011)

Another option that is being researched is the use of optical sensors. These allow non-invasive sampling through a transparent window. These sensors can be coupled to a disposable system but the systems themselves are reusable. ( Eibi R 2011)

5.13 Centrifugation

This is usually one of the first steps performed after fermentation or cell culture to remove cells and cell debris from the solution before it is then moved on for downstream processing. Due to this complexity there has been difficulty in designing an effective single use system at the moment there are only two different manufacturers producing these and they are KSep Systems and Carr Centrifuges. These two manufacturers work along different principles. KSep systems is based on a number of revolving chambers fitted with one disposable bag, this allows the procedure to be carried out in a closed system and can be used on volumes from 1- 6000 litres . Carr Centrifuges is based on a tubular bowl centrifuge that is then lined with a disposable bag this can be used on volumes up to 1000 litres. (Shukla A 2012)

A study has been carried out where a centrifuge was replaced with a disposable depth filter in the clarification of a Chinese Hamster Ovary cell culture. The cell culture clarification was originally carried out by an initial centrifugation step followed by a single layer depth filter and then a 0.2um membrane filter. A 1000 litre batch was processed without the centrifugation step, the batch went straight to the single layer depth filter and then through a 0.2um membrane filter. It was found that the removal of the centrifugation step had no effect on the filtrate that was produced for downstream processing and allowed for an easy and convenient set up and a significantly reduced cleaning time for equipment and the process suite. (O Brien T.P 2012)

5.14 Depth Filters

Along with centrifugation, depth filters can be used after fermentation or cell culture in a clarification step designed to remove the cells, cell debris and other assorted matter from the solution in order to produce a particle free solution that can be more easily managed in the downstream process. Traditional depth filters are usually made up of cellulose fibres and various filters aids that are then bound by a polymeric resin which strengthens the material. Initially they are produced in flat sheets; these sheets are then assembled into multistack housings. A typical large scale depth filtration system uses multistack housings with up to 16 inch depth- filter modules which are stacked three or four high in order to increase the surface area. The void volume and particle retention rating of a filter depends on how well the fibres are compacted together and on the length of the fibres. The tighter the particles are bound together the better the retention of the particles. (O Brien T.P 2012)

Disposable depth filters are among the most commonly used disposable technology in biopharmaceutical production. There are a number of different companies that supply them including Millipore, Pall, Cuno and Satorius-Stedium. (Shukla A 2012). Millipores have created the Pod filter system. This is a modular designed depth filter which comes in seven filter sizes and with a choice of two expandable holders and can be used for volumes from 5 to over 12000 litres. ( http://www.millipore.com/catalogue/module/c9485). Pall has developed a system called Stax. These are 3 different size capsules that range in size from 0.25M2 to 25M2 which can then be placed into one of three different size chassis and can be used for volumes from less than one litre to greater than 20,000liters. (http://www.pall.com/main/biopharmaceuticals/product.page?id=49738)

A Study has been carried out to compare a depth filter using stainless steel housing with one that is disposable. A two-step stainless steel housing based "half thickness, dual layer media" was replaced with a disposable EZP capsule with "full thickness, dual layer" depth filter technology in the processing of 10,000 litres of whole Chinese Hamster Ovary cell culture. These full thickness, dual layer depth filters are increasingly being used in biopharmaceutical production, they are produced by layering two sheets of filter media of differing pore sizes and then assembling this into lenticular style cartridges. With these systems a more open filter is placed upstream to remove an large cells and debris and a smaller filter is placed downstream to remove any finer particles, by aligning these three filter sizes it allows for a greater depth filter capacity and an reduces the damages that may occur to downstream membranes .

In the case study carried out there it was seen that there was a number of advantages to using the disposable system over the traditional stainless steel one including time and operator safety. When the traditional system is being used it takes approximately nine to ten hours from set up to completion however when the disposable system was used the process was complete in approximately 3 hours and there was a much easier set up as there was no need for winches or other equipment that would be needed with traditional systems to open the stainless steel housings. The disposable system was also designed to allow easier confirmation that the filters were in place and there was no need to install gaskets as there would be with the stainless steel system. The process itself was also improved as the transparent cover of the disposable system allowed the operators to see what was happening in the filter allowing them to monitor the process efficiently this was especially relevant during the blowdown and postflush phases. The disposable system was also set up to allow easy venting of the core of the filters which is not possible with the stainless steel systems, this venting is useful as it prevents air from reaching the downstream membrane and eliminates the need for prolonged venting of a membrane to prevent it air locking. There were also a number of advantages with regard to operator safety as the system was ergonomically designed to allow loading and unloading at waist level. (O Brien T.P 2012)

5.15 Virus Clearance

There are a number of regulatory guidelines on the removal of virus from biopharmaceuticals. The EMEA outlines a number of different characteristics that potential virus contaminants may have including being enveloped or non-enveloped, small or large, deoxyribonucleic acid or ribonucleic acid, unstable or resistant. Q5 of the ICH guidelines mandates that manufacturers of therapeutic biological products for human use implement adequate technologies in there manufacturing process and demonstrate the capability of their processes to remove or inactivate known or adventitious contaminants based on a process specific virus clearance strategy. For a manufacturer to be able to licence their product they must be show viral safety by the use of three complementary approaches including a throughout testing for virus contaminants of the cell line and all of the raw materials used in the manufacture, assessment of the capacity of the downstream process to clear any infectious materials that may enter the process and finally ensuring that the product is tested at appropriate steps throughout the process for viral contamination. When designing a process to ensure that their product will be free from viral contamination the manufacturer can use viral removal or viral inactivation. Methods for viral removal include chromatography steps such as size exclusion or ion exchange or filtration steps whereas viral inactivation can be carried out by pH changes, solvents or detergents, microwave heating or chemical treatment. When trying to move to a disposable system for removing or inactivating viruses manufacturers have been looking toward the chromatography or filtration step in the process. With regard to chromatography systems s