Equipment and Scaleup

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The availability of hardware at the required scale, or the complexity of its deployment, may influence which techniques are used and how they are implemented.

Large-scale engineering, for example of flow distributors, can be a critical issue. The difficulties of large-scale operation can be balanced against the cost of running multiple cycles at a

Cell Culture Equipment

Figure 18.3 Pilot-scale columns used in Phase II clinical manufacture. A 30-litre size exclusion column and a 2-litre ion exchange column used for production of clinical lots for a protein vaccine product at Xenova. In these Millipore Vantage A columns, the headspace above the top adjustable adapter can be pressurised to enable rapid and consistent column packing. Photo courtesy of Jim Mills, reproduced with permission of Xenova.

Figure 18.3 Pilot-scale columns used in Phase II clinical manufacture. A 30-litre size exclusion column and a 2-litre ion exchange column used for production of clinical lots for a protein vaccine product at Xenova. In these Millipore Vantage A columns, the headspace above the top adjustable adapter can be pressurised to enable rapid and consistent column packing. Photo courtesy of Jim Mills, reproduced with permission of Xenova.

smaller scale. This approach is often used for size exclusion, but as the technique is slow, multiple cycles may take days to complete and this in turn will impact on the economics of the facility. This technique can be difficult to implement at commercial scale, and scale-up can cause a loss of the required resolution (see Section 18.5.5).

The selection of chromatography hardware will be influenced by the scale of operation. Columns of up to about 100 l such as the BioProcess Glass columns from GE Healthcare, can be packed using traditional hardware. Other column ranges offer zero dead space around the adapter seals, such as the Vantage columns from Millipore (Figure 18.3), and these are potentially more easily cleaned or sanitized. Such columns are also available with air packing, which allows the column to be packed by applying air pressure in the headspace above the adjustable adapter.

Columns of 500 l or more can be custom made from stainless steel. At this scale, it is very costly to have adjustable adaptors, but it is very difficult to pack columns of the correct bed volume using fixed headplates, and to do so without headspace voids.

Column Packing
Figure 18.4 A large-scale, 1.5m diameter Chromaflow column, when used with a semi-automated packing station, enables clean and efficient column packing at large scale. Photo courtesy of GE Healthcare. Biosciences AB

Chromaflow columns, from GE Healthcare Bio-Sciences AB, offer a semi-automatic method of packing that can be of benefit when packing large columns of up to 900 l in a consistent manner (Figure 18.4). The columns are packed by pumping a matrix slurry into a fixed column tube. These columns do not require adjustable adaptors, and the column tube is made to the required length to accommodate the required bed volume. These columns can also be unpacked without dismantling the column hardware, which can be an advantage in minimizing production downtime for column packing.

A similar packing method is used in radial-flow columns, e.g. from Sepragen. In radial-flow columns, the flow is applied to the column around the circumference of the column tube and liquid flows through the matrix towards the centre of the column where outflow is collected along the axis at the centre of the column. This is different to axial-flow columns where flow is applied to the top cross section, flow is parallel to the column axis, and the outflow is collected at the bottom cross section.

Axial column scale-up is generally achieved by maintaining the linear flow rate and the column bed height, whilst increasing the cross-sectional area and volumetric flow rate. In an axial flow column, the linear flow rate is constant along the flow path.

Radial-flow columns are scaled up simply by increasing the length of the column tube and the volumetric flow rate, and this minimizes the footprint required by the column at manufacturing scale. However, the linear and volumetric flow rates across the radial flow path are not constant, and the flow rate increases as the flow nears the centre of the column.

It is necessary to ensure that scale-up of the process will be possible (Sofer & Hagel 1997). The commercial scale will influence how the process will work; for example any operations involving

Cell Culture Process Development
Figure 18.5 A 1/10-scale heat exchanger used in the development of a process to cool 50L of clarified harvest in the production of a live virus vaccine. Photo courtesy of Jim Mills, reproduced with permission of Xenova.

mixing, changes in temperature, or low temperatures, will be more difficult, or take longer, the larger the scale.

It is important to attempt to model the anticipated large-scale equipment capability during development. For example, in a particular process requiring the cooling of 500 l of clarified product for the manufacture of a live-virus vaccine, a heat exchanger had to be employed to reduce the temperature from 34 °C in the culture vessel to 8 °C, within one hour, for the subsequent downstream process step. A 1/10-scale process was used in development and, rather than rely on variable cooling in a refrigerator, the manufacturer identified for the large-scale heat exchanger supplied a 1/10-scale model to achieve the same cooling in the same time for 50 l of harvest. The 1/10-scale heat exchanger is shown in Figure 18.5. The implementation in the laboratory of this scaled-down model of the full-scale equipment ensured that scale-up would not change the process.

The stainless steel employed for process hardware, buffer tanks or pipework needs to conform to the relevant standards, for example the FDA regulation 21 CFR 211.65 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'; further information can be found at http://www.fda.gov/cder/dmpq/cgmpregs.html. 316L stainless steel is accepted in the industry as the standard for any metal in contact with product or process fluids. The design of the steelwork needs to be sanitary and cleanable, for example with no dead-legs and with smooth flow paths (see Chapter 14).

It is an increasing expectation of regulatory agencies that manufacturers develop good working relationships with suppliers of equipment (and raw materials), and it is commonplace for suppliers to provide information to the manufacturer for equipment validation, for installation and operation qualification, and this will help minimize the work required of the manufacturer and optimize the validation process for cGMP purposes.

Disposables are increasingly used in manufacture to avoid the difficulties of cleaning and cleaning validation. Polypropylene is a common non-leaching plastic used for bioprocess containers. Disposable flexible bags or plastic liners for tanks in volumes up to about 1500 l, such as those from combrex, Sartorius, HyClone and Stedim, are now commonly used for large volumes of buffers or product handling. It is important to assess disposables for leaching of chemicals, such as plasticizers and anti-oxidants. For example, water for injection with a known total organic carbon (TOC) content can be placed in a flexible bag and sampled at intervals for TOC analysis. Leaching may occur, in the order of thousands of ppm, and the water may fail a <500 ppm TOC limit within a few days. It may be required by regulatory authorities to test leaching from bioprocess containers in contact with the process fluids, such as buffers and column eluates, under the conditions used in the process. It is possible that the plastic film itself does not leach under conditions that comply with the USP Class VI test, but plasticizers may be liberated when the disposable product is gamma-irradiated and a build up of TOC may be present when the bag is used.

As stated before, it is important to consider the large-scale implementation of the process during development, such as the types of column, UF membrane or disposables to be used, so that changes introduced later in the project are minimized. For example, the need to repeat toxicology studies can arise if new product-contact materials are used that have the potential to leach different amounts or types of components into the product, and this will inevitably cause costly delays to a project.

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