A new method for the manufacture of powdered cell culture media

1st April 2013

Due to the continuing growth of the biopharmaceutical industry, in recent years, there is an increasing demand for large volumes of both powdered and liquid media for production purposes. Alan Baines reports on a new method to manufacture a powdered cell culture media which overcomes many of the disadvantages of previous methods.

Many biopharmaceutical customers are using cell culture media as a raw material for the production of human therapeutics from mammalian cell culture. As the regulatory environment for the manufacture of biopharmaceuticals is necessarily strict, there is a now greater requirement than ever before, for media manufactured to meet current industry standards.

While it is possible to make liquid media from scratch formulation i.e. from the individual components, increasingly both media manufacturers and the bioprocessing industry, rely on ready-to-use powdered formulations.

During 1995 BioWhittaker introduced a novel and innovative process to produce powdered media of a higher quality than possible with traditional methods. This process, known as micronisation, was first introduced in BioWhittaker's facility in Walkersville, USA. Starting in late 1999 BioWhittaker's manufacturing unit in Verviers, Belgium has constructed a completely new and innovative micronisation facility for production of powdered cell culture media for its European customers.

Traditional method

The traditional method of manufacturing powdered cell culture media is to use a process known as aball milling'. In this process the raw chemicals for the specific formulation are introduced into a ceramic lined container and a number of ceramic balls, known as the acharge', are added. The container is then sealed and rotated to allow the charge, over a period of time, to pulverise the raw materials with the objective of producing a fine powder.

The powder is then sieved to separate it from the charge before being passed to a blending cone or ribbon to allow mixing of sequential batches produced by the milling process which can only handle relatively small lot sizes.

This method whilst now in use for many years suffers from a number of key disadvantages which affect both powder quality and cause some regulatory problems for the end user.

Among the key disadvantages are:

1. Production of significant quantities of heat which may damage key media components reducing cellular growth and protein production capability. This problem has been frequently masked in the past by the necessity to add serum, which by its very nature has variable growth promotion capabilities. This is much more likely to be a problem with the new serum-free formulations.

2. Generation of ceramic dust and fragments in the end product, which may clog filters and/or enter the final product. It is not unknown for a ball from the charge to end up in the powder shipped to the end user.

3. The process depends on human intervention due to the staged nature of the production. It is difficult therefore, to avoid human contact with the material during the milling and blending process thus increasing the risk of contamination.

4. The process is time consuming, as multiple lots are needed from the milling process to make a single large batch of powder, thus increasing the risk of error in the final formulation. It is also difficult to respond rapidly to customer demand.

5. The cleaning process is also slow and very difficult to validate, yielding risk to the end user of contamination between formulations.

6. Control of humidity during manufacture can be very difficult and some manufacturers are forced to introduce drying processes to reduce the water content. This also increases the risk of damaging key components in the final product.

7. The combination of points 4, 5 and 6 lead to long lead times for the production of specific formulations, incompatible with the increasing requirement for ajust-in-time' supply requirements.

It was these considerations, several of which have potentially serious implications for biopharmaceutical manufacturers that led BioWhittaker to develop the micronisation process. The new facility being completed in Verviers incorporates several new developments to the original concept, offering European customers the highest possible quality and regulatory assurances.

The process of manufacturing high quality powdered cell culture media incorporates several key stages, which have all been carefully addressed by the Verviers production team. The end result promises to be raise the industry standard for this type of application.

Raw materials

Inevitably the selection and qualification of raw materials is an essential part of the initial process. In order to ensure that the starting materials are of the highest quality and traceability, audited suppliers capable of meeting manufacturing requirements, must be assembled.

In recent years concerns have grown over the use of components of animal origin due to the incidence of retrovirus contamination and the T0SE problem. We now have a comprehensive database, assembled and vetted over several years by experienced members of the purchasing team. This database contains information from numerous suppliers such as the grade of material EP, USP, etc, purity, non-animal origin status, likely endotoxin levels etc. This provides the Verviers facility with rapid access to the correct material to meet the customers specific needs. The latest EC resolutions also obliged us to take into account the need for provision in some cases, of aCertificates of Suitability'.

Identification and weighing

Customers and the regulatory authorities are now looking for incontrovertible evidence that the formulation contains the stated chemical components at the specified purity. In order to provide this vital evidence, Verviers has installed the latest Near InfraRed spectroscopic analyser to guarantee chemical integrity.

This unit is capable of analysing an unknown sample, comparing it to a stored database of identified components of specified purity and indicating the nature and acceptability of the material. This allows the Verviers facility to provide customers with traceability for raw materials even down to the provision of the printouts of the NIR analysis data when requested.

A new set of segregated weighing stations has been installed to enable the rapid, traceable assembly of the raw material components for each formulation. Each weighing station is actually an individual suite, with its own aDust Hog', which assists in preventing cross-contamination during the assembly of raw materials.

Environmental controls

The production of powdered media ideally needs to be in a clean, dry environment. The hygroscopic nature of many of the components and the final product, mean that a low relative humidity is needed to avoid problems with clogging, weight gain of components etc. Most ball milling facilities have limited capability to control humidity and at least one manufacturer still uses ambient humidity conditions. The resulting powder created under these less-than-optimum conditions must then be dried before it can be packaged and sold. The drying process also involves heat, creating conditions that may favour degradation of components, or even worse, bacterial growth.

The Verviers facility's original design incorporated segregated air handling though out the manufacturing plant. We have taken advantage of this capability to create a powder-manufacturing environment with remarkable humidity control. In our facility we can actually create a relative humidity of as low as 12 per cent, but this would make the working environment inhospitable for the operators involved. We therefore use a specification of 25 per cent relative humidity, which in layman's terms could be compared, to the Sahara desert at high noon. We are able to maintain this low relative humidity level throughout the entire powder-manufacturing unit. This is possible even when during cleaning cycles are carried out in adjacent rooms. This means that the resulting powder is produced in optimum conditions during the entire process and does not need any corrective drying cycle. Powder produced by traditional ball milling methods frequently needs a special drying cycle, where the powder has to be subjected to a higher temperature. This undesirable step in the traditional production cycle, threatens component integrity and overall performance characteristics.

Dust control

The production of a powder inevitably produces quantities of powder that are not captured during the process, known as adust'. This unwanted dust then has the potential to cross-contaminate subsequent production runs, if it is not dealt with properly.

Dust is a constant problem for producers of powdered cell culture media, especially those using the old-fashioned ball milling process. It is notable that when Pharmaceutical companies are invited to audit such facilities, it is most likely to be a astage audit', that is offered by the manufacturers using the ball milling process.

The micronisation process allows a much better control over the process and the generation of adust'. The Verviers facility uses automatic pneumatic transfer from the microniser to the blender and from the blender to the filling machine. Unlike the ball milling process, no human intervention is necessary. As a result significantly less dust is created and the use of specially designed adust-hogs' at the filling stations minimises any possibility of cross-contamination from dust particles.

The manufacturing process

Micronisation uses a special modification of the ajet-milling' process used for a number of years already in the pharmaceutical industry. The raw chemicals are introduced into the microniser, which contains a stainless steel rotor, revolving at high speed. The raw chemicals impact the rotor and are instantly pulverised. The resulting fine powder is then conveyed pneumatically to a double cone blender. Unlike ball milling, this is a continuous flow process and theoretically there is no limit to the batch size that can be produced by this method. Practically, however, the size of the blender limits the final batch size. BioWhittaker with both its Verviers and Walkersville facilities can manufacture batch sizes from as small as 400 litres up to 500 000 litres, depending on the density of the formulation. This provides potential customers with a complete scale up path from initial pilot scale operation to full-scale production, using the same process.

The specifications and characteristics of the BioWhittaker microniser, regrettably must remain proprietary but there are a number of important factors which should be mentioned as they influence the quality of the end product.

1. The most important is particle size. Lack of uniformity in particle size may compromise the blending process, leading to a non-homogenous product. If on the other hand, the process yields a range of particles that are too coarse, dissolution will be slow and the solubility of key components may be adversely affected. This ultimately will reduce the growth promotion properties of the final product. Conversely, if the powder is too fine, the powder will also prove difficult to dissolve with some important components afloating' on the surface during rehydration or worse, drifting away into the surrounding atmosphere.

2. Lack of heat build-up. The grinding action involved in ball milling generates heat over a long period of time. It is known that some components are adversely affected by heat and this may result in sub-optimum performance from the final formulation. Heat is of course, also generated by the impact during micronisation, but this is momentary and quickly dissipated. Growth promotion tests seem to indicate that micronised powdered media outperforms that produced by the traditional methods.

3. Ease of cleaning. BioWhittaker has for the first time, established a full cleaning validation for the simpactor surfaces. Due to the nature of ball milling, components are ground into the ceramic surfaces of both the charge and the mill. Validation of cleaning procedures is therefore made extremely difficult. The use of only pharmaceutical grade stainless steel within the simpactor, allows full validation of the cleaning process.

4. Large-scale biopharmaceutical production is expensive and there is constant pressure to reduce the costs of all aspects of the production process, including raw materials. The micronisation process provides sufficient improvements over the original methods to offer end users, high quality raw materials at very competitive costs.

The final part of the process, filling the relevant containers for the powder, is also carried out under optimum conditions using a calibrated filling machine. Throughout the manufacturing process the trained operators wear a combination of maximum-security pharmaceutical gowning and where relevant, special isolator helmets. This means the operators can be completely isolated from contact with the powder at any stage, especially during critical stages such as weighing of the raw materials and dispensing final product. Opportunities for cross-contamination are thereby minimised.


BioWhittaker Europe has constructed a new facility for the production of high quality, powdered cell culture media. This unique European facility uses innovative technology in the design of a production process that avoids a number of significant disadvantages, inherent in the old-fashioned ball milling technique. The micronisation method allows much more rapid response to urgent customer demands as both the manufacturing cycles and the cleaning cycles are considerably shorter than with the ball milling method. Such flexibility can be of considerable advantage to the biopharmaceutical industry. The resultant powdered media is ideally suited to biopharmaceutical industry demands for raw materials, that improve regulatory compliance and ease of production, whilst keeping raw material costs as low as possible. Micronisation heralds a new era for the production of powdered cell culture media for biopharmaceutical customers throughout the world.


Alan Baines is the co-ordinating director, sales and marketing with BioWhittaker Europe, Verviers, Belgium.





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