from contamination. Vials are cleaned using ultrasonic baths filled with cleaning solutions to remove any particulate debris prior to sterilisation. The outer and inner surfaces of the vial undergo further rinsing with fresh wash solution and then purified water to remove traces of the cleaning solution. This is followed by a drying step with a jet of sterile air. The cleaning and rinsing solutions, and the air used for drying, are all passed through 0.2 Î¼m filters to remove any microbes and particulates before contacting the vials.
Figure 1: Example of an ultra-sonic cleaning machine for glass vials. It contains pumps and filters to circulate the cleaning solution (taken from Kambert, 2009).
Pyrogens are inactivated rather than removed from vials by the use of dry heat. The vials pass through an oven whilst sterile filtered air is passed through in the opposite direction. This means that the vials are cooled upon leaving the oven by the in-flowing cooler, filtered air (see figure 2). Other methods to inactivate pyrogens include ionizing radiation, oxidation and chemical inactivation (Williams, 2007). Thermal exposure of glass vials can cause delamination, but fortunately depyrogenation does not cause delamination to occur; it is more likely during a terminal sterilisation step (Iacocca et al., 2010).
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Figure 2: Scheme of a depyrogenation oven. The vials are placed on the conveyer belt and pass through the oven from left to right. The filtered air flows in a counter current direction from right to left.
Vial filling with liquids uses specific vial filling devices. The final product is passed through a 0.2 Î¼m filter to remove any microbes just before entering the filling apparatus. There are several methods to aliquot the required amount of solution into each vial. Conventional systems use either a piston or diaphragm pumps, or a time-pressure system (Belongia et al., 2002). Diaphragm and piston pumps are most accurate when new as their accuracy relies upon the quality of their manufactured parts. Pump wear will compromise accuracy and could lead to particulate contamination. Time-pressure controlled systems have time-controlled valves and hold tanks pressurised by nitrogen. The constant pressure of the solution maintains its flow rate. Although simple, they can suffer from variability at lower fill volumes (Belongia et al., 2002). Systems that fill by weight can be more accurate as the weight of the individual vial is constantly measured during filling. In these mass-flow systems a feedback loop operates, stopping the filling once the desired amount is obtained. These systems are not appropriate for viscous solutions, but are often used for ophthalmic solutions (Heyman, 2009).
Figure 3: Vial filling equipment. The vials are on a conveyer belt that passes below the fill head needles. A time-pressure system is used to aliquot the solution into the vials (taken from API, 2010).
After filling with the drug product, vials are inspected for signs of damage or contamination. Inspection devices specific for detecting and counting particulates are used (Walsh, 2007). These are based on the transmittance of light through the filled glass vial (Tatford et al., 2004). The selectivity of this method is further enhanced by the use of motion. By spinning the container and stopping it quickly, any particulates in the liquid will continue to move, whilst marks on the container itself will remain still. This method is generally used for closed carpoule liquid fill product presentations. Lyophilised powder formulations will undergo inspection to ensure a uniform cake is obtained during the lyophilisation process. All presentations are inspected to ensure cap and vial integrity is maintained throughout the capping process.
Figure 4: Scheme of a particle detector. The light is focused by a lens and passes through the sample vial. After focusing by a second lens it hits a detector. Any particles present will scatter the light beam and prevent it from reaching the detector.
Describe, with the aid of your own diagrams and sketches, a suitable facility layout for a vial filling process.
When designing a suitable facility for vial filling the nature of the final product needs to be considered. In this scenario, the mAb preparation will be in solution, but will require lyophilising once in the vial so that it is supplied as a dry powder requiring reconstitution just prior to administration. This is to ensure its stability. Sterilisation via heating is the preferred method for sterile products (Walsh, 2007). However, for heat labile products such as the mAb, sterilisation via filtration and the use of aseptic processes are employed instead.
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The vial filling facility is designed according to the individual steps in the process: the receipt of the materials, weighing, filling, lyophilising, capping and sealing, and then shipment. By viewing the whole process, the movement of equipment, substances and personnel are arranged as efficiently as possible (Dahlmanns, 2009). There are four cleanroom classes and passing from one zone to another involves standardised decontamination processes. The design (see figure 5) means that the necessity of passing through air-lock doors is minimised so as to increase efficiency and reduce the contamination risk. The filling machine is U-shaped to reduce the space needed in the highest grade clean-room. A single employee in the filling room monitors the process, and this reduction in activity in the clean-room reduces the risk of contamination (Lysfjord, 2008). A continuous flow of sterile air from inside to outside of the clean-rooms and container handling operated by fixed installation gloves, minimise contamination. The operator and product are always physically separated, where possible. The filling and sealing is performed in the highest category clean-room, with the final capping of the vials in a separate zone. This is due to the capping procedure generating particulate matter that could contaminate the product (Flaum, 2006).
Figure 5: Floor plan of the vial filling facility. The clean rooms are divided into different zones with zone A being the cleanest and zone D the least clean.
A restricted barrier access system (RABS) is used (see figure 6) that segregates operators from product. This reduces the exposure of the pharmaceutical to viable organisms or particulates (Lysfjord, 2008). These systems use high efficiency particulate air filters that supply air conforming to the ISO 5 standard. The isolation of the filling line increases the level of assurance, and the cost savings offset the initial cost (Schreier et al., 2009). The air handling systems use a positive flow as purified air enters the system and flows out into the room. Devices placed in the air exit flow can monitor the cleanliness of the system. These systems meet the regulatory standards, are flexible and easily customised to individual processes making them ideal for the production of low volume high value biologics such as the mAb (Schreier et al., 2009). These protein-based drugs require highly aseptic conditions as they are often preservative free and, through their production involving growth media, are easily contaminated (Lysfjord, 2008).
Figure 6: An example of a RABS system used to produce sterile powders for pharmaceutical use (Dahlmanns, 2009).
The clean room environment is maintained by the use of Heating, Ventilation and Air Condition (HVAC) system that uses positive pressure to maintain sterility (Stockdale, 2004). The design of the building and rooms, the HVAC and air-handling system, disposal systems for contaminated waste and the movement of operators will all affect the efficiency of a process (Lysfjord, 2008). Consideration of personnel flow is crucial to maintaining the sterile environment. Following gowning their movement should be from cleanest area towards the less clean areas, where possible. The processing equipment is positioned so as to allow routine maintenance to be undertaken without the need for entry into the clean area as far as is possible. The mechanics and electronics are physically isolated from the parts that come into contact with the drug product and vials.
Part 3: Formulation Goals
When setting the formulation goals the desirable properties of the product are mapped:
Physicochemical and biological properties
The drug product physiochemical properties are evaluated. These include its:
The goal of the formulation is to maximise the efficacy of the drug product. As therapeutic proteins have limited stability, certain additives will need to be included in the formulation, and it will be lyophilised to increase its shelf-life.
The goals of adding excipients are to:
Enhance the stability of the drug product
Enhance its activity
Improve its pharmacokinetics
The stability is improved by excipients that can give a buffered pH of the correct value. In addition, those preparations that are being delivered straight into the body intravenously, subcutaneously or intramuscularly, should be of physiological pH and ionic strength. Excipients will also stabilise the protein against agitation, drying and freezing (as when lyophilised). They include fillers, diluents, wetting agents, solvents, emulsifiers and preservatives (see below).
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The lyophilisation process may cause aggregation of the drug product as the water content is reduced. Excipients that offer protection during the freeze drying process include buffers, sugars, amino acids, surfactants and preservatives. The buffer must be chosen carefully so that any crystallisation of the buffer salts during the lyophilisation process will not cause a detrimental pH change to the remaining protein solution.
The concentration of drug product will increase as it is dried and, given that the starting concentration may be high, means that aggregation could be problematic (Salinas et al., 2010). Sugars can act as osmotic protectants, but reducing sugars such as maltose cannot be used with protein drug product due to the disulphide bonds it contains. Therefore the stabilisers sucrose or trehalose could be used.
The aim of the bulking agents is to improve the physical properties of the freeze-dried protein. A denser solid is formed that has better dissolution properties (Bedu-Addo, 2004). This is important so that the end user does not end up with a non-uniform suspension after reconstituting it. This would affect the accuracy of the dose and be problematic to dispense.
All of the excipients must be compatible with the active ingredient and the manufacturing process. As protein drug product is sensitive to heat will be sterilised via filtration, excipients must be highly soluble, pure, sterile and not cause foaming.
The goal of any adjuvant is to increase the immune response to mAbs. Adjuvants that are used in conjunction with mAbs include aluminium salts, Freund's adjuvant (FA) and squalene (Chen et al., 1993). By increasing the body's reaction to the mAb they can help lower the amount required to elicit a response. This reduction in the effective dose can help reduce the size of the volume to be injected, making it better for the patient. This is particularly pertinent to mAbs; they often require high doses to be effective (Salinas et al., 2010). In addition, carriers such as keyhole limpet hemocyanin (KLH) can also be used to increase the efficacy of mAbs (Chen et al., 1993).
Preservatives are added to the drug product with the goal of maintaining its structural integrity and as a result its physiological function (Walsh, 2007). For example the mAb may undergo reduction of its disulphide bonds that will cause its structure break down. In a contrary manner, oxidation of redox sensitive side-chains could also reduce its activity if they play a role in its interactions with other proteins in the body. Preservatives that prevent such oxidation include methionine, ascorbic acid and butylated hydroxyanisole.
In addition, preservatives are added with the goal of preventing microbial degradation of the antibody and harmful infection of the patient. Antimicrobials are added to maintain sterility and prevent any microbial growth. These include compounds such as phenol and 2-phenoxyethanol. However, single dose formulations manufactured under sterile aseptic conditions such as the mAb here may not need an antimicrobial adding. If it is required, it should be added at the lowest level at which it is effective due to its inherent toxicity.
Container closure systems
The container closure system will allow the sterility and integrity of the drug product to be maintained (Bhamra et al., 2000). It will allow easy visual detection of any tampering and enable the product to be used easily when required.
The drug product will be packed in glass vials. These can be stoppered prior to lyophilising and then sealed afterwards. Teflon-coated stoppers are preferable so as to minimise the adherence of lyophilised protein to the stopper. The seal will create a tamper proof closure and add an additional layer of safety by displaying the instructions "dilute before use".
Dosage forms and delivery systems
Agents such as the drug product that are being delivered by injection must be sterile (Bhamra et al., 2000). Avoidance of contamination, be it microbiological, particulate or pyrogenic, is necessary. Therefore, the delivery system must allow aseptic production and the maintenance of the sterile environment. A single use vial for a single injection is the preferred option (Stokowski, 2012).
The goal of the dosage forms is to reduce the need for frequent repeat injections. Increasing the time the drug is circulating in the bloodstream can do this. PEGylation of the drug product will reduce the rate of clearance via the kidneys (Chapman, 2002). PEGylation will reduce the rate of proteolysis and the immunogenicity of the product. It will also increase its solubility and enhance its stability. An assessment must be made of the suitability of PEGylation for mAb as some antibodies are incompatible with the chemical polymer.