The beginning of the end

There are many challenges upstream and downstream in manufacturing a biotech drug. Research on the upstream side in recent years has produced gene expression systems that have allowed for dramatic increases in yields of monoclonal antibodies, new vaccines and recombinant proteins. Downstream developments are catching up, with products such as the disposable Rhobust column from Upfront Chromatography (Denmark) and GE Healthcare's PreDictor, which is a 96-well plate system for rapid testing of purification conditions such as pH and ionic strength. However, purification is not the end of manufacture — it is just the beginning of the end.

Susan Aldridge

The main challenge of getting a biotech drug from this stage to the patient is the delicate and unpredictable nature of protein molecules. According to Geoff Helleswell of Micropharm (UK), the manufacture of their anti-sera and polyclonal antibody products takes a couple of days, but formulation, filling and signing off a batch can take 4–6 weeks. The cost of formulation and filling, as well as ensuring stability, is a big issue for the company as it produces antivenoms for countries, such as Nigeria, that cannot afford expensive freeze drying, which is a popular formulation technology for protein drugs.

"There are several stages involved in the formulation and filling of a biotech drug", says Helleswell. Some of them are similar to those for small molecule drugs, but regulatory authorities often demand more data for a protein drug because of its inherent instability, and various tests must be done on the bulk product, with bioburden (microbial content), protein concentration, and virus removal or inactivation being the most important.

Sterilization

At this stage, the question arises as to whether the product can be terminally sterilized. For a biologic, the answer is usually 'no' as protein molecules unravel when exposed to the heat of a sterilization operation. Therefore, filtration into a previously sterilized container will be necessary, with a recommended second filtration step immediately prior to filling. "Double filtration is very important for biologics," says Helleswell.

At this stage, the product is being exposed to the air so consideration must be given to the environmental conditions for filling. "The environment in which the filling is done is critical," explains Helleswell. An EU Grade C environment is fine if terminal sterilization is intended, but the higher Grade A is necessary if the product has to be filled under aseptic conditions, meaning that manufacturers, including CMOs, must invest in the appropriate clean room technology if they are going to do their own formulation and filling.

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Most biopharmaceutical products are injected. This means preparing either a freeze-dried solid product that is reconstituted in the clinic or solutions that are contained in either capped vials or sealed ampoules. Prefilled syringes, for ease of patient use, are an increasingly attractive alternative in some applications. Additives, such as salts or preservatives, might be necessary to stabilize the protein and keep it stable for whatever storage times and conditions are appropriate to the final end use. A great deal of development work is needed to select the most appropriate additives to ensure they do not give any untoward clinical side-effects.

Inspection

The filled product must be inspected for complete closure and for visible and subvisible contamination. Meanwhile, sterility is checked with samples that are representative of the whole batch. Other tests, such as protein concentration, pH, osmolality and activity may also be required.

Visual inspection, which can be performed by eye by a validated operator or with an automated visual inspection machine using lasers, is an important check. Regulatory authorities have requirements for clarity and place limits on even subvisible particles in a product. Proteins tend to precipitate, adsorb onto surfaces, or aggregate — all of which can cause visible or invisible deposits.

The clinical significance of aggregation processes — particularly whether they can cause unwanted immune side-effects — is currently a subject of some debate. No protein product can be guaranteed to be 100% free of aggregation, but it should be understood and controlled as much as possible for a particular process and product. Aggregation often goes undetected and the underlying causes can be quite complex. pH and ionic strength of the formulation mix tend to be important factors and freeze drying often causes aggregation in some products when they are reconstituted. Aggregation can also occur during transport of a product by the use of pumps and even certain types of packaging. Additionally, shaking a protein solution is less likely to cause aggregation than stirring it.

Visual inspections are important for assessing formulation stability as well as protein aggregation. There are a number of approaches for assessing the particle content of a protein solution. Light extinction methods are used to work out the actual size of the particle, but they are not applicable if the particle is translucent. Sometimes the sample must be diluted, which can cause interfering air bubbles, and may, in itself, cause aggregation.

Detecting aggregation

Experts in bioprocessing use a variety of methods to detect the degree of aggregation in a particular product, which goes from the simple monomer to a visible aggregate particle; size exclusion chromatography remains the standard, although dynamic light scattering (DLS) and analytical ultracentrifugation (AUC) are often used. DLS is good for examining larger particles, while AUC is well suited to resolving a mixture of particles of different sizes and can give a handy chromatogram-like result. However, neither method is easy to use or validate for batch sign off purposes. As regulatory authorities expect manufacturers to use more than one approach to investigate products, a combination of these technologies is being increasingly used in formulation development.

One new approach that can be used in this context is Micro Flow Imaging (MFI), developed by Brightwell Technologies (Canada), which combines the speed of a particle-counting method with the visual insight of a manual microscopic approach. In MFI, a volumetric section of the protein solution is investigated so particles are counted, sized and imaged. It allows all kinds of particles, protein aggregate or not, to be measured and assessed. Protein aggregates can be tricky to study because they are fragile, highly transparent with irregular size and shape, but MFI can cope with these issues. In an example presented at a recent bioprocessing conference (Vienna, Austria; April, 2008) by the company, MFI was shown to be highly efficient in checking the effectiveness of a filtration step in particle removal, and causing (or not causing) aggregation to occur. In other experiments, MFI could detect particles formed when a protein solution was forced through a syringe.

Once the batch has been formulated, it is on to the final stages — and the paperwork. Helleswell describes the subsequent labelling and packaging steps as a "minefield". Labels must be authorized by regulatory authorities and special attention paid to their position, particularly if they are being applied manually as is often the case if costs are to be driven down. Then, the final product must be assembled, complete with the patient information leaflet. The packaging must ensure that the product stays in good condition during storage and transport, which is more challenging for a biotech product than a small molecule.

Then there is the paperwork (increasingly being done in paperless format). A batch manufacturing record (BMR) covers all records during production, labelling and packaging and quality control for inspection by the Qualified Person (QP). The QP certification includes review of the BMR, production conditions, stability reports for the expiry date, validation, GMP standards required and authorization/licences available. Afterwards, the product is finally shipped and distributed, but to complete the life cycle there must be some arrangement for complaints, returns and disposal to be dealt with.

Many of the steps described are common to conventional small molecule drugs and biologics, but there are differences because most biologics drugs are proteins, which vary from batch to batch in a way that a synthetic chemical does not (or should not). This means that formulation and the final product should be considered early in manufacturing so that the potential heterogeneity of the batch itself — and between batches — is better understood and controlled. Helleswell says: "In general, biological medicinal products require enhanced in-process controls to ensure batch-to-batch consistency."