Change of culture

BSE has had massive public health implications. Here Phil Smith of Oxoid looks at its impact on pharmaceutical manufacture and at how companies can avoid prion contamination.

'Upon conclusion of the process we hit the "Prion Destruction" button'. Nice idea, isn't it? Pushing a magic button would certainly solve the dilemma many people face when handling products that have components of an animal origin.Until that day arrives, manufacturers face a difficult challenge in balancing product safety and quality, with guidelines and regulations, alongside the constraints imposed by current technologies.

Most people will be familiar with 'Mad Cow Disease', better defined as Bovine Spongiform Encephalopathy (BSE). The disease first appeared in the UK in 1985, when cattle began to show symptoms of nervous collapse. The following year, the cause was linked to animal feed containing ruminant remnants (cattle, sheep, goats) or ruminant by-products. Despite this knowledge, the offending feed was not strictly regulated and BSE continued to spread.

In 1996, the probable link between eating contaminated beef, and developing a neuro-degenerative disease in humans was made.¹ Even today, the agent responsible for BSE has not been fully characterised. However, the probable cause is currently accepted to be prions.

unusual agents

Prion is shorthand for proteinaceous infectious particle and many people assume prions to be similar to pathogenic micro-organisms, but this is far from the truth. They are unusual as infectious agents as they are devoid of any nucleic acids, being composed solely of protein.² Normal cellular glycoproteins (PrPC) are converted into abnormal, 'infectious' forms (PrPSc).

It is now accepted that transmission from animals to humans can occur, hence the classification as Transmis-sible Spongiform Encephalopathies (TSEs). Prion diseases have been shown experimentally and clinically to be transmitted via contaminated surfaces or materials of an animal/human origin.³ Generally, prion diseases produce neuro-degenerative symptoms, such as dementia and lack of co-ordination, and are always followed by death.

tough measures

Since 1986, almost every European country slowly began to confirm cases of BSE, and tough measures were introduced in an attempt to halt the spread of the disease. To date, around 150 people have developed the human form, nvCJD, in the UK and most patients will die within 12 months of presenting with the disease. Japan recently confirmed its first nvCJD death, Canada confirmed Chronic Wasting Disease in a deer,⁴ and the US is reeling with confirmation of its second BSE infected cow.⁵

At present, there is no vaccine, antidote or cure for any prion disease. They are among the most fascinating and alarming diseases facing us today. Allowing prions to contaminate parenteral or infusion products could prove fatal.⁶ The problem is global.

Bovine materials are widely used in biopharmaceutical production, medical device manufacturing and reprocessing facilities, plasma fractionation and dietary supplement manufacturing. Any of these manufacturing processes that use (or could potentially use) or contact tissues of animal or human origin, have a potential, although considered very low risk, of prion contamination.⁷ This contamination could be transferred into the pharmaceutical product. Such contamination is unacceptable and taken extremely seriously.

structure precautions

Companies take very structured precautions to prevent unwanted contamination of their products. This includes the installation and running of critical areas (e.g. cleanrooms), rugged environmental monitoring programmes and the use of strict decontamination procedures and validated cleaning processes.

Raw materials are often treated to reduce the risk of microbial contamination. Liquids are filtered to remove particulates and micro-organisms, containers are autoclaved, medical devices may be irradiated. Despite all of this, prion contamination could arise from the use of production and validation materials that contain components of human and animal origin.

Areas of concern include in vitro diagnostic devices, culture media for biopharmaceutical production/validation and blood/plasma fractionation.

The risk of prion contamination is highest with neural tissues, including that of the brain, spinal cord and optic nerve.⁸ Neural tissues create a cleaning challenge, as they are difficult to remove from surfaces due to their high lipid content. Prions, in particular, strongly adhere to stainless steel surfaces. Even with good cleaning, removal alone is not the answer, as it potentially moves the prion contamination from one area to another, without actually inactivating the abnormal protein.

The inactivation of prions is an extremely difficult task. Numerous studies have shown that no single method of decontamination is completely effective. A mixture of methods is used, including cleaning followed by chemical decontamination and possibly steam sterilisation.⁹

risk recognised

Prions are more stable than anthrax spores; they are not inactivated by irradiation, and, worryingly, no routine measures exist to detect them in situ. Both the World Health Organisation (WHO) and Federal Drug Administration (FDA) have recognised the risk of using bovine materials in the preparation and manufacture of parenteral and infusion products. Materials have been classified into categories of increasing infectivity risk.

In 2001 and 2002, the FDA recommended that the risks of prion contamination should be minimised. Since the discovery of the 'mad Canadian cow' in the US in 2003, and subsequent American cases, the US regulatory authorities have increased their diligence in this area. Despite this, more cases are being identified across the globe.

Guidance asks manufacturers to adopt cleaning, disinfection and sterilisation methods to reduce the risk of prion cross-contamination, and use bovine materials only where necessary, and source such products from 'BSE-free' countries.¹⁰

Although there have been no reported cases of BSE transmission from vaccines and other products, failure to comply with manufacturing regulatory guidelines can lead to the costly withdrawal of products. Under 1999 European Guidelines,¹¹ an oral polio vaccine was recalled in 2000, upon the discovery that a growth medium containing UK bovine material was used in the vaccine's production. The downturn in public confidence, subsequent damage to the company's reputation and financial impact was very noticeable.

Manufacturers of biological products are asked to supply information on the sourcing and control of animal substances used in the manufacture of a product, as well as supplier information to assure and document the health and origin of the animals used. Manufacturers are also required to supply detailed information on cell culture history, isolation, media, identity and testing of cell lines used in the production of biological products.

With all of this theoretical risk, manufacturers are obliged to scrutinise every aspect of their process, and provide documented evidence of compliance with the regulations. Active Pharmaceutical Ingredient (API) suppliers to the pharmaceutical and related regulated industries are also obliged to follow tight restrictions regarding the sourcing of animal component materials. These suppliers must provide evidence to demonstrate thorough sourcing, regulation and certification of raw materials, purchasing bovine materials only from 'BSE free' countries. However, is perfectly reasonable to assume that countries certified as‚ 'BSE-free' today, could identify cases in the future.

In September 2004, after 17 years, the FDA updated its guidance and released 'Guidance for Industry - Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practices'. The world was a very different place when the original guidance was issued; 'Mad Cow' disease was a relatively unknown problem and the new guidance was updated to reflect current needs and technologies.

One of the most obvious changes is the increased focus on ensuring the sterility of products produced in aseptic conditions. Manufacturers were familiar with using microbiological culture media as a substitute for their products in process simulations (media fills). The risk of microbial contamination could be assessed from such exercises.

The new guidance places more emphasis on these process simulations, with manufacturers using more culture media, more frequently. Such media is often Tryptone Soya Broth, and, as such, contains components of animal origin. In doing this, the microbiological risk is being dealt with, but the risk of prion contamination has theoretically increased.

In response to these issues, some culture media manufacturers have taken several approaches towards providing animal component-free products, where possible supplanting bovine and other animal raw materials with vegetable-based products. Any product containing vegetable-derived material would have to demonstrate equivalent performance characteristics in order to be an attractive and viable alternative to the traditional formulations for the pharmaceutical industry.

At Oxoid, considerable time was spent designing a vegetable based growth media for use in microbiological and fermentation processes. A vegetable protein source (pea flour) is used, along with bacterial and fungal proteolytic enzymes (which replace the more traditional trypsin and porcine pancreatin enzymes). As an additional secure step, the enzymes used are sourced from micro-organisms cultured on bovine-free media.

viable alternative

Vegetable Peptone Broth or Veggietone Broth was designed as a viable alternative to Tryptone Soya Broth. Used as a general purpose medium, the Veggie-tone broth demonstrated comparable microbiological performance to Tryptone Soya Broth when recovering low numbers of cells. For increased security, the components of the vegetable peptone products are certified as free from genetically modified (GM) materials. These products have undergone microbiological quality control testing according to the guidelines set out in US, British, European and Japanese Pharmacopoeia.

Some of the world's largest pharmaceutical manufacturers are exploring animal component-free alternatives for their critical processes. Until we find the 'magic button', the use of a high quality vegetable-based product, that does not undermine but supports the pharmaceutical process, is a credible step to take.