Protein Formulation

Background

Protein therapeutics which occupy about 13.8% of the global market are biological drugs that can be used for treating cancer and other infectious diseases. Monoclonal antibodies are a great example of biologic drugs which are used in immunotherapy because of their high target specificity and lower side effects (1). They are usually administrated parentally because of their big molecular size and hydrophilicity which could lead to a poor bioavailability if administrated orally (2). Protein therapeutics are sensitive and demands a specific formulation as well as processing design in order to maintain their primary, secondary, tertiary and quaternary structure. Often the tertiary structure which is retained by noncovalent interactions characterise the biological activity of a protein and preserves stability as well as toxicity outcomes. Therefore, protein structural maintenance is important during formulation for a maximal therapeutic effect and avoidance of aggregation which can leads to immunogenicity (3). Immunogenicity is the body s immune response against therapeutic protein like monoclonal antibody which results in the generation of anti-drug antibodies that deactivate the therapeutic effect of a treatment and causing adverse reaction in patients (4).

Immunogenicity is an immune response of the body to pathogens that occur as a result of protein aggregation, precipitation and denaturation of the therapeutic protein, when these reactions occur, the product is degraded. Therefore, protein stability is a key factor that should be considered for a successful formulation (5). Aggregation (figure 1) is a serious biological issue which could lead to precipitation. It happens as a result of accumulation of misfolded protein intra or extracellularly because of hydrophobic interactions between the proteins molecules (6). Many factors such as storage temperature, pH, excipients and high concentration could promote aggregation. Therefore, it is important to maintain the right storage temperature, physiological pH (7-7.4) and adding suitable excipients to the formulation that can increase the solubility in addition to the stability of the protein. This essay will be focusing on factors that promote aggregation and the techniques that could be used to overcome this biological phenomenon.

Figure1. An illustration of protein aggregation, precipitation, denaturation and adsorption.

Pre-Formulation Consideration

It is essential to understand the properties of a protein prior formulation for designing and developing a suitable formulation process. Table 1. Highlights some of the key parameters that should be considered during the pre-formulation stage (7).

Structural Parameters

Physical Parameters

Biological Parameters

Primary

Secondary

Tertiary

Quaternary

Solubility

Hydrophobicity

Molecular size/ weight

Self-association

Aggregation state

Glycosylation

Substrate affinity

Receptor affinity

Biological activity

Pharmacokinetic profile

Table 1. Pre-formulation characterisation parameters for therapeutic proteins(7).

Many factors such as pH, temperature, isoelectric points and hydrophobicity affect solubility which governs the therapeutic activity of a protein. This is because proteins tend to self-associate and aggregate at certain pH level and temperature which causes the therapeutic protein to un-fold (denatures) and loses its function. The intermolecular interactions among proteins and glycan moieties results in Glycosylation, which could results in the unfolding of the quaternary and the tertiary structure. When this process happens protein loses its function. Also, it is important to evaluate the biological parameters like the substrate affinity, receptor affinity of a formulation as they govern bioavailability and its therapeutic effect. The creation of pharmacokinetic profile of a protein in terms of absorption, distribution, metabolism and excursion is one of the vital requirements for predicting the biological activity of a formulation. This data can be done through mass spectroscopy based techniques as well as imaging tools which helps in choosing suitable excipients for a successful formulations of a biological dosage forms (7).

General Requirements for Therapeutic Protein Formulation

Biological drugs demand a different route of manufacturing process than the conventional pharmaceutical products due to their structural stability that should be maintained for its therapeutic effect. The manufacturing process should comply with the Good Manufacturing Practices and maintains appropriate conditions such as sterility. This means the design of manufacturing process should contain no contaminations from dust, fumes, etc. Thus, product sterilisation is an important requirement for the safety and efficacy of the final dosage form, and since proteins are sensitive to heat, the autoclaving method should be avoided. Instead, a mechanical technique like filtration should be considered for any bacterial removal (8).

The typical protein shelf-life is about 18 months so it is essential to provide the right storage temperature <4 ^oC, to maintain the product stability, avoid aggregation, precipitation and denaturation during storage. Additionally, maintaining a physiological pH (7-7.4) is important for improving patient s compliances and avoidance of pain during injection. Therefore, adding suitable buffer that can maintain a physiological pH and other excipients to increase the solubility are important during the development of protein formulation. Due to the sensitivity of the proteins to factors like temperature, pH, pressure and tonicity, stability studies should be performed under stresses like light, humidity, refrigerators as well as agitators. This is to establish the product stability during transport and handling as well as storage (8).

Choice of container

Dark opaque glass I is considered to be the ideal container choice for therapeutic protein because of its inertness. The choice of the elastomeric closure system is critical as rubber can interact with the polymeric excipients of the formulation and cause immunogenicity. The selection of the elastomeric closure is dependent upon several aspects like the drug itself, the buffer in addition to the pH, preservative, sterilisation methods as well as the humidity protection. Hence, the choice of the container is as critical as the formulation process. This is because the selection of the wrong container type such as polystyrene or with plasticizer coating could lead to aggregation which causes immunogenicity (9).

Excipients

Excipients are generally added to increase the product solubility, stability and in return improving the products shelf-life by minimising the risk of aggregation and precipitation. Therefore, the choice of suitable excipients is critical during the formulation process. The use of multifunctional excipients are highly favoured in protein formulation. For instance, if an excipient has a buffering capacity while at the same time is capable of improving the solubility and the stability, it will be considered an ideal choice (10). Table2. Provides a list of excipients that could be added to protein therapeutic formulation for improving the solubility, stability and minimising the risk of aggregation (11).

Excipients

Function

Examples

Buffers

Maintain pH level and stabilises the formulation upon adding an acidic or a basic compound.

Acetate, phosphate, glycine, and citrate.

Sugars and polyols

Protect the folded state/the tertiary structure of protein against temperature and chemical denaturants by providing higher hydration. A minimum of 5% is recommended in a formulation.

Glucose, sorbitol, lactose, ascorbic acid.

Salts

Reduces Protein s electrostatic interaction, enhancing solvent s activity and stabilises protein. Salt is protein dependant and may or may not be effective.

Sodium Chloride(NaCl) stabilises protein like IL-1R.

Potassium Chloride, KCl

Polymers

Improve protein stability by non-covalent interaction. The stabilisation is determined by the type of protein and the size of PEGs.

Polythylene glycols (PEGs) is commonly used in protein stabilisation. The molecular weight of the PEGs ranges from 100-1000 Da.

Solubility-enhancers

Increasing the solubility of proteins.

Amino acids, detergents and surfactants like poly-sorbate 20 and 80.

Anti-adsorbent and aggregation blockers

Reduce of adsorption and prevent aggregations.

Albumin detergents, surfactants

Table 2. A list of some excipients that could be added to a protein formulation (11).

Freeze-drying

Liquid therapeutic protein are susceptible to aggregation and degradation because of the presence of water which promote chemical and physical instabilities (12). Freeze drying is a process that could be used to improve the protein s stability during storage. The process involves removal of water by sublimation. There are three stages involves in this process which includes freezing, primary drying and secondary drying (figures2,3). However, this process is harsh and has the potential to introduce physical instabilities such as denaturation of the therapeutic protein (13). To minimise the risk of physical instabilities excipients like bulking agents, suitable buffers for pH as well as osmotic adjustment, cryo-protectants, protein structure stabilizers, and even phase-state modifiers which is the glass transition temperature modifiers are added to increase the physical stability of the protein (14,15). Generally, a low concentration of the protein is used in the formulation due to its great efficacy and this could contribute to the potential loss its therapeutic effect during freeze-drying (14). To minimise the risk of protein damage bulking agents like lactose, glycine, sucrose and mannitol are added to increase the bulk mass of the formulation. These excipients can also be used as cryo-protectants (protein stabilizers during freeze drying and storage) to optimise the collapse temperature and by this means improving the stability of a formulation (15). The collapse temperature is associated with the product temperature and is used to measure the ideal temperature of the storage (16). The Following (figures 2,3)represent 3 stages of freeze drying (17).

Figure2. Different stages of the freeze drying process (17).

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Figure3. Three stages of the freeze drying cycle (18).

Techniques That Could be Used to Minimise Protein Aggregation

Parental formulations are susceptible to aggregation which can cause unwanted immunogenicity therefore, they have to be formulated with some specific considerations to minimise the risk of aggregation and maintaining the therapeutic effect throughout the product shelf-life. Generally, there are some guidelines that could be followed to minimise this biological phenomenon which include, maintaining low protein concentration as high concentration could increase the molecular interaction of protein and cause aggregation. However, if the product demand a high protein concentration then consider using solubility enhancers such as Amino acids or polysorbate 20/80 to reduce the molecular interaction of protein and increase the solubility of protein formulation. The usage of the multifunctional excipients is a great advantages to the formulation as it reduces the amount of excipients being used in a formulation. This is reflected in the product performance as well as the manufacturing cost. Consider the freeze drying technique and add excipients like cryo-protectant to maintain the stability during the process. Finally, proteins are heat sensitive so storing them at the <4^oC is an advantageous for improving the product shelf-life (19).

The Importance of Formulation in Lifecycle Management

Life cycle management (LCM) is an important business strategy that is used by the innovator companies to prevent the generic companies entering the market and maintaining product exclusivity. This is crucial for maximising the profit margin of the innovator companies. Thus, the innovator companies should come up with a bio-better version of a formulation like New formulation, New Indication, New product presentation or New combinations to extend the patent of a formulation. Exploring one of these technique is sufficient to stop the generic companies entering the market and extending the patent life of a product (20). In order to attain new patent duration or extend the product patent, the FDA requires a proof that demonstrate the new formulation is a bio-better version of the old formulation which is beneficial to patients. For instance, Avonex powder form was the first generation of the Avonex pen which had to be diluted prior injection (figure 4), (21). This demonstrate the importance of the product presentation in the patent extension of the product. Therefore, from the economical point of view it is essential for the innovator companies to keep coming up with a bio-better version of the product for patent extension in order to eliminate the generic companies entering the market.

Figure4. The three generations of AVONEX (21).

Conclusion

In conclusion, Protein formulation could be a challenging process so the formulation scientist should have a strong background in protein chemistry, physical pharmacy and formulation principles as well as a strong team of bio-analytics for an effective formulation development. Therapeutic protein are sensitive to factors such as heat, pH, choice of the container and can easily lose their structural integrity which lead to protein denaturation, aggregation or precipitation. Therefore, one should consider these factors carefully and formulate under appropriate conditions to overcome the unwanted immunogenicity which causes the body to have adverse reactions. In conclusion, biologic drugs demand a different formulation process and conditions than the conventional formulations in order to maintain their 3-dimentional structure which determines its biological activity.