Formulation and Instability of Biotechnology Products

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By Alison Symington, PhD

The development of pharmaceutical products made through the use of biotechnology has significantly affected the development of processes for purification, formulation and stabilization. In order to discuss this class of products, it is necessary to define them.

What is a biotechnology product? Biotechnology is defined by the Canadian government as “the use of living organisms or parts of living organisms to improve the quality of human life.” Alternatively, biotechnology can be defined as biological answers to everyday problems.

Biopharmaceuticals (also known as biotechnologicals) are defined as products made through the use of recombinant technology, where genetic manipulation of cells is required. Biopharmaceuticals differ from traditional biologics in that biologics are directly purified from their original biological source. Biologics include blood products, traditional vaccines and hormones, to name but a few. The discussion that follows will not deal specifically with these products, but with recombinant DNA products. That being said, many of the issues that surround formulation of recombinant products also affect the purification and formulation of biologics as well.

Currently, nearly all approved biopharmaceutical products are recombinant-protein-based. Due to the nature of the proteins, these products are sterile, parenteral products. They are usually purer than their biological equivalents and are closer to the true biological product than a synthetic pharmaceutical. The basic production process for all recombinant protein biopharmaceuticals is the same. A recombinant organism (usually E. coli or a eukaryotic cell line) is transformed to harbour the necessary piece of DNA.

Following propagation up to commercial scale, the protein product of the gene must be isolated and purified. Typically, after a crude protein broth is concentrated, high-resolution chromatography is used. The protein products from these processes are usually at least 95% pure, given today’s advanced purification technologies. The final phase of processing involves formulation of the final product. It is at this point that formulation becomes an issue.

Impurities

The first issue that affects formulation is the constituents of the final product. If a final product is only 95% pure, it contains significant amounts of impurities, which can lead to either a more stable or, in fact, a less stable product. As with synthetic pharmaceutical products, impurities that cannot be removed must be consistently present, not toxic and show no biological activity. Interestingly, if in fact the impurities lend a measure of stability to the product, it may be that formulation of the intended product must account for instability of those impurities.

The profile of impurities between a synthetic pharmaceutical and a biopharmaceutical are significantly different. Impurities in a synthetic pharmaceutical product tend to originate from the process itself, involving such things as solvents or isomeric products. In a biopharmaceutical process, the majority of the product impurities are derived from the host itself and not introduced into the process. The development of procedures to deal with these impurities derives from both the current literature and from regulatory guidance. The growth of biopharmaceuticals has mirrored the development of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance documents. There are several essential guidance documents that are relevant to the purification and formulation issues surrounding a biopharmaceutical process (Table 1).

There are also additional guidelines in the Q2 series regarding testing and assay validation. The issues regarding the formulation of biopharmaceutical products can be broken down into sensitivity to sterilization, and more importantly, the intrinsic instability of products made through biotechnology.

Sterilization Issues

Proteins by their very nature are sensitive to a variety of environmental conditions. Terminal heat sterilization, which is commonly used with synthetic pharmaceuticals, cannot be used with proteins. Protein products may be irreversibly denatured through the heating procedure. As with all small molecules, structure and function in these products is integrally related. Denaturation of a protein will destroy its function and therefore its biological activity. Typically, filter sterilization is used for recombinant products. The largest problem encountered with filter sterilization is protein aggregation. Ionic manipulation of the solution or the addition of anti-chelating excipients can reduce this problem.

Intrinsic Instability

The very nature of the recombinant product makes it unstable. In order to understand the parameters of your product, evaluation of the stability of the product will need to take a multifaceted approach. All testing methods need to be related to biological activity as that is truly the measure by which your product will be judged. Very often, multiple tests may need to be done to assure that biological activity has been retained.

In order to judge the stability of the protein product, a reference standard needs to be generated. Unlike most synthetic products, such protein products will not have a USP reference standard. The standard will be a “professed,” or in-house, standard, subject to the same issues as your product. In order to ensure that the reference standard is relevant, it should be clinically relevant. That is, the product should be shown to be effective biologically in the clinic. Typically, a proportion of a lot that has demonstrated success in the clinic can be quarantined to act as a reference. As the development process moves through Phase 1 to Phase 3, it is quite likely that the reference standard will change as more information is available. All following reference standards can then be measured against this “gold standard.” The ICH guideline Q5C is particularly useful at this point. Typically these references are kept frozen and it is necessary to perform a complete stability study on your reference standard as it is performed on your product.

Instability of a recombinant protein can be separated into either physical instability issues or chemical instability issues. Physical instability can be related to such things as denaturation of the secondary and tertiary structure of the protein; adsorption of the protein onto interfaces or excipients; and aggregation and precipitation of the protein.

In most biopharmaceutical processes, additives are used to improve the physical stability of a protein. The addition of salts can significantly decrease denaturation and aggregation by the selective binding of ions to the protein. Polyalcohols can also be used to stabilize the protein by selective solvation. Finally, surfactants are often used to prevent the adsorption of proteins at the surface, although there is a fine line between the amount of surfactant needed to prevent adsorption and the amount needed to denature a product. In addition, excipients are often used to prevent aggregation. However, many excipients are derived from animal sources. These sources are problematic as there are specific quality procedures that need to be followed to assure that these raw materials are free of such contaminants as BSE. In multivalent products, such as multivalent vaccines, excipients are not always compatible with all the active ingredients. As with all process development, the more that is known about the characteristics of the protein, the greater the chance to develop a robust product.

Chemical instability of a protein product results in the formation of a new chemical entity by cleavage or by new bond formation. Examples of this type of instability would be deamidation, proteolysis and racemization. There are some more obvious choices to improve the chemical instability, such as modulation of pH, the use of low temperatures for storage and processing, and the addition of preservatives. Additionally, proteins can be chemically modified so that they are coupled with such chemicals as polyethylene glycol or lipids, which can promote the absorption of the protein.

Chemical stability can also be affected at the gene level. Through the process of site-directed mutagenesis, the chemically reactive amino acids can be replaced with less reactive ones. This procedure can be cumbersome and of course relies on the integrity of the protein product remaining intact. However, if it is possible, site-directed mutagenesis removes many of the issues during downstream processing and final formulation of the product.

When is Instability Not a Problem?

Intuitively we know that instability of a protein is a problem, but there are cases where too much analysis is done, allowing for interpretations that are not meaningful and not related to the ultimate goal. Many biological products have structural heterogeneity due to the biosynthetic processes the organism has used to make the protein. Many times, the final broth may be a mixture of anticipated post-translationally modified forms. All of the forms may be biologically active.

The issue here, as put succinctly at the Toronto, Ont.-held Joint Calibration & Validation Group/Therapeutic Products Directorate 2003 International Convention in late September, is that differences are only important if they are substantially different. What does this mean? For many products it is the dreaded case-by-case evaluation. But in reality what this means is, if you can show that the heterogeneity is anticipated, consistent and has no effect on the biological activity of the product, then these differences are not substantial and minimal time should be spent on evaluating them.

Alison Symington, PhD is a professor at the School of Biological Sciences and Applied Chemistry, Seneca College of Applied Arts and Technology (Toronto, ON).