Biopharmaceutical proteins: biophysical studies of aggregation and their interactions with excipients

Traditionally, drugs used to treat disease have been small molecules- these are relatively simple chemicals, perhaps consisting of fewer than a couple of hundred atoms. They are sufficiently small to be made by direct chemical synthesis. Although this type of drug is still widely used, more recently a different kind of medicine has been introduced. These drugs are much larger molecules- containing many thousands of atoms- and are generally proteins. They have significant advantages over small molecule medicines, and have extended the scope for treatment of disease. Therapeutic antibodies are a good example: they have been developed to treat a range of human diseases, including multiple sclerosis, autoimmune diseases and cancer. The method of manufacture of these drugs- or biopharmaceuticals- is different from their small molecule counterparts, however. They are too complex to be made by direct chemical synthesis; instead, they are frequently made from cultured mammalian cells.

The use of this new kind of drug has raised its own types of problems. Large proteins are generally much less stable to certain conditions, such as heat, compared to their small molecule counterparts. Considerable effort is therefore expended by the manufacturers for each new biopharmaceutical medicine to find conditions under which it is stable, and thus prolong its 'shelf life'. A major source of deterioration in stored preparations of biopharmaceutical drugs is aggregation- this is a technical term which describes the propensity for proteins to bind to each other in solution. Aggregates are effectively 'clumps' of protein which are many times larger than the original protein, and form spontaneously under certain conditions. Boiling an egg induces a similar kind of behaviour- the egg white protein (albumin) is pulled apart by the elevated temperature and the individual protein chains adhere into a dense mesh. This becomes insoluble, which is why the albumin is transformed from a transparent, colourless liquid into a white solid. The presence of aggregates in the preparation of a biopharmaceutical drug can be deleterious to its function- it can reduce its efficacy and also, on some occasions, induce the production of antibodies in the patient in response to its administration.

One way to reduce aggregation is to add certain small molecules- called 'excipients'- which have the effect of stabilising the biopharmaceutical drug. The way in which excipients function is complicated, however, and not well understood. This proposal seeks to investigate how they function, using a range of different experimental tools which are designed to measure the behaviour and structure of proteins in solution. We will conduct these measurements under different conditions and with selected excipients which are commonly used. The information we obtain will be used as a practical guide to help in stabilization of new biopharmaceutical drugs, as they are developed in the future. We will also develop specific methods which can be used to provide a practical guide during drug development. The project is a partnership between an academic grouping, who have expertise in the various methods employed, and MedImmune, who have extensive experience in developing these new kinds of medicine and bringing them to market.

The project will examine effects of solution conditions and small molecule excipients on protein folding stability and dynamics, protein self-association and the link to aggregation through:

1. Determination of excipient effects on protein self-association and interactions between partially folded states by light scattering and computation:
a) Measurement of native protein self-association as a function of ionic strength and pH. Electrostatic calculations will be correlated with experimental measurements at low ionic strength to determine the role of net charge and charge distribution.
b) Examination of the effect of selected excipients and salt/buffer types on native protein self-association.
c) Protein-excipient interactions: computational tools under development will be applied to the study of excipient binding, and comparisons made with experimental measurements.
d) Extension to partially unfolded forms: influence of partial denaturation on protein self-association and its modulation by excipients.

2. Investigation of the link between weak protein-protein interactions, protein unfolding, aggregation kinetics and concentrated solution behaviour, using light scattering and image correlation spectroscopy methods.
a) High throughput identification of melting and aggregation onset temperatures.
b) Mechanistic studies of aggregation behaviour.
c) Solubility, mutagenesis and patches: computational methods will be used to identify charged surface residues for mutation and experimental analysis.

3. Studies of the binding and effects of excipients on the structure and dynamics of small antibody fragments.
a) NMR studies to examine the effects of selected excipients on dynamics using i) standard heteronuclear relaxation parameters (R1, R2 and NOE) ii) H/D exchange
b) Identification of excipient binding sites by X-ray crystallography

1. The Pharmaceutical and Biotechnology Industries
An obvious primary non-academic grouping who would benefit from this research are bioprocessing specialists, specifically those involved in formulation, and biochemical engineers. The project will provide information which will help to uncover the principles by which aggregation occurs and how this is modulated by certain excipients. The project has its most obvious impact in formulation- the last step in the development pipeline for biopharmaceuticals. The work also impinges on development work earlier in the pipeline- during protein production and purification. There are clear advantages in the use of small molecules to stabilize protein products during certain steps in the purification process, for example, and reduce losses from irreversible aggregation. Specific goals are included within the Pathways to Impact plan, to enable engagement between the academic applicants and biopharmaceutical companies.

In addition to bioprocessing specialists, the project will also be of benefit to biochemical engineers i.e. those interested in and working with industrial processes involving proteins and protein-related products. It is important to emphasize that the scope of this impact extends well outside healthcare products and biologics. The production of enzymes for other industrial processes- the manufacture of fine chemicals for example- also relies on stabilization and improved shelf life of proteins.

2. Health and Medicine Regulatory Bodies
The importance of aggregation in biologics is still a matter of active debate, but it is an area of increasing concern to bodies involved in regulating biological medicines. Aggregates span a very wide range of sizes, from nm up to sub-visible and visible particles of micron dimensions. Of principal concern here is the role which aggregates could play in reducing drug efficacy or, in extreme cases, even pathology in the patient. Aggregates are known to have the potential to induce an immunogenic response, even if the administered drug has been 'humanized' i.e. aggregate formation is able to break self-tolerance mechanisms. How this occurs is not entirely clear at present, but the production of anti-drug antibodies has been demonstrated, and is a phenomenon which can reduce the potency of the drug. Clearly, therefore, the relationship between aggregation and the excipients included in drug formulation is a matter of importance for regulators. Joint meetings will be held with members of regulatory bodies, to disseminate the results of this research.

3. Patients who will be treated with biopharmaceutical medicines
Finally, the most important group who will indirectly benefit from this project are patients. They will benefit in three ways. First, more efficient means for stabilizing biologics, reducing losses during production and more effective formulations should contribute to lowering costs, benefitting the Health Service and taxpayers more generally. More robust formulations will also offer alternative options for transport and storage: for example, the removal of cold shipping requirements with 'room temperature stable' biologics. Second, as outlined above, a reduction in aggregation in an administered biologic will have health benefits for the patient, by improving drug efficacy (by reducing anti-drug antibodies) and improving safety (by reducing still further the probability of more serious consequences arising from an immunogenic response to drug aggregates). Third, the ability to produce liquid formulations of antibodies at ever higher concentrations will have an immediate impact on drug administration: for example, it could help to switch the need for a subcutaneous (s/c) syringe pump or multiple s/c injections to the delivery of a larger dose in a single injection.