Formulation Approaches for Biologics: Strategies to Enhance Stability and Bioavailability

Richard Saraiva^*
*Correspondence: Richard Saraiva, Department of Pharmacy and Research, University of Michigan, 500 S State St, Ann Arbor, MI 48109, USA, Email:

Keywords

Posterior • Biologics • Bioavailability

Introduction

Biologics, including therapeutic proteins, monoclonal antibodies and vaccines, represent a significant advancement in modern medicine due to their ability to target complex diseases with high specificity. However, their formulation presents unique challenges related to stability and bioavailability. Biologics are sensitive to environmental conditions and can degrade or lose efficacy if not properly formulated. Addressing these challenges requires innovative strategies to maintain their stability during storage and improve their absorption in the body. This article explores various formulation approaches designed to enhance the stability and bioavailability of biologics, highlighting recent advancements and their impact on therapeutic outcomes.

Literature Review

Biologics are prone to degradation through various mechanisms such as aggregation, oxidation, deamidation and hydrolysis. These processes can result in loss of biological activity and formation of potentially immunogenic particles.

Stability is a critical concern in the formulation of biologics, as their complex structures make them susceptible to various degradation mechanisms. Addressing these stability issues is essential for ensuring the safety, efficacy and shelf life of biologic products. Key stability challenges include:

Aggregation occurs when biologic molecules clump together, forming larger particles or aggregates. This process can be triggered by factors such as protein concentration, temperature fluctuations, or changes in pH. Aggregates may not only lead to a loss of biological activity but can also induce immune responses in patients [1].

• Mechanism: Aggregation can be caused by hydrophobic interactions, electrostatic forces, or conformational changes in the protein structure.
• Impact: Aggregates may lead to reduced therapeutic efficacy and increased risk of adverse immune reactions.

Oxidation involves the modification of amino acid side chains, particularly methionine and tryptophan, which can alter the protein’s structure and function. Oxidative degradation can be exacerbated by exposure to oxygen, light, or metal ions.

• Mechanism: Oxidation can lead to changes in protein structure, affecting its stability and activity.
• Impact: Oxidized proteins may lose their therapeutic activity and become immunogenic.

Deamidation is the process where asparagine or glutamine residues in a protein undergo chemical modification to form aspartic acid or glutamic acid. This reaction is influenced by pH, temperature and time [2].

• Mechanism: Deamidation can cause changes in protein charge and conformation, impacting protein stability.
• Impact: Deamidated proteins may exhibit altered activity or stability, potentially leading to reduced therapeutic efficacy.

Hydrolysis involves the cleavage of peptide bonds within a protein, typically driven by water and catalyzed by acidic or basic conditions. This process can lead to loss of protein structure and function.

• Mechanism: Hydrolysis can degrade proteins into smaller fragments, which may have reduced or altered biological activity.
• Impact: Hydrolyzed proteins may lose their intended therapeutic effects and contribute to the formation of immunogenic peptides.

Denaturation refers to the loss of a protein’s native structure, often caused by temperature extremes, pH changes, or shear forces. Denatured proteins can aggregate, further complicating stability issues [3].

• Mechanism: Denatured proteins may expose hydrophobic regions that lead to aggregation and loss of functionality.
• Impact: Denatured proteins can result in reduced efficacy and potential immunogenicity.

Biologics are often sensitive to temperature changes, requiring strict temperature control during storage and handling. Elevated temperatures can accelerate degradation processes, while freezing can lead to protein aggregation.

• Mechanism: Temperature fluctuations can impact protein structure and stability, leading to accelerated degradation.
• Impact: Poor temperature control can compromise the integrity and efficacy of biologic products.

The stability of biologics can be significantly affected by pH levels. Deviations from the optimal pH range can lead to protein denaturation and aggregation.

• Mechanism: pH changes can alter protein charge and structure, affecting solubility and stability [4].
• Impact: Proteins may become less soluble or more prone to aggregation under non-optimal pH conditions.

Bioavailability of biologics can be hindered by factors such as poor solubility, instability in the gastrointestinal tract and inefficient absorption across biological membranes.

Bioavailability refers to the proportion of a nutrient or medication that enters the bloodstream when introduced into the body and is thus available for use or storage. It is a critical factor in determining the efficacy of nutrients or drugs [5].

1. Formulation and administration:

• Oral vs. Intravenous: Oral medications often have lower bioavailability due to first-pass metabolism in the liver.
• Solubility: Poorly soluble drugs have reduced absorption in the gastrointestinal tract.

2. Chemical properties:

• Molecular size and polarity: Larger or highly polar molecules may have difficulty crossing cell membranes.
• Stability: Some compounds may degrade before absorption (e.g., in the acidic environment of the stomach).

3. Physiological factors:

• Gastrointestinal pH: Variations in pH can affect drug solubility and stability.
• Gut flora: The microbiome can metabolize certain compounds, affecting their absorption and bioavailability.
• Food interactions: Certain foods can enhance or inhibit absorption (e.g., grapefruit juice affecting drug metabolism).

4. Metabolic factors:

• First-pass metabolism: Drugs absorbed through the gastrointestinal tract must pass through the liver, where they may be metabolized before reaching systemic circulation.
• Enzymatic activity: Variations in enzyme levels (e.g., cytochrome P450 enzymes) can affect drug metabolism.

5. Transport mechanisms:

• Passive diffusion: Lipophilic compounds typically pass through cell membranes more easily.
• Active transport: Some nutrients or drugs require specific transport proteins to cross cell membranes.

Strategies to enhance stability

• Glycosylation modification: Altering glycosylation patterns can enhance protein stability and reduce immunogenicity [6].
• Stabilizing mutations: Site-directed mutagenesis to introduce stabilizing mutations can increase resistance to environmental stressors.
• Stabilizers: Common stabilizers include sugars (e.g., trehalose, sucrose) and amino acids (e.g., arginine) which help to maintain protein structure during lyophilization and storage.
• Preservatives: Preservatives such as benzyl alcohol or phenol prevent microbial growth in formulations, extending shelf life.
• Liposomes and nanoparticles: Encapsulation in liposomes or nanoparticles can protect biologics from degradation and enhance their stability.
• Microencapsulation: This technique involves encasing biologics in a polymer matrix to protect them from environmental conditions.
• PEGylation: Conjugation with polyethylene glycol (PEG) increases molecular size, reducing renal clearance and extending circulation time.
• Extended-release formulations: Formulations designed for slow release can improve bioavailability and patient compliance.
• Subcutaneous and intramuscular injections: Alternative routes can enhance absorption compared to intravenous administration.
• Oral delivery systems: Techniques such as enteric coating and absorption enhancers aim to improve the bioavailability of biologics administered orally.
• Permeation enhancers: Compounds that temporarily increase permeability of biological membranes to facilitate better absorption.
• Nanotechnology: Utilizing nanoparticles to transport biologics across cellular barriers more effectively.

Recent advancements in monoclonal antibody formulations include the use of Fc-fusion proteins and improved stabilizer systems to enhance stability and efficacy.

Discussion

Definition: Monoclonal antibodies (mAbs) are laboratory-produced molecules engineered to serve as substitute antibodies that can restore, enhance, or mimic the immune system's attack on cells. They are specifically designed to bind to antigens, which are unique proteins on the surface of cells, including cancer cells, viruses, or bacteria.

1. Antigen Identification:
• The first step involves identifying a suitable antigen that the monoclonal antibody will target.
2. Immunization:
• A mouse or other suitable host is immunized with the antigen to stimulate the production of antibodies against it.
3. Cell fusion:
• Spleen cells from the immunized host are fused with myeloma cells (a type of cancer cell) to create hybridoma cells. These cells can produce antibodies and have the ability to proliferate indefinitely.
4. Screening and selection:
• Hybridoma cells are screened to identify those producing the desired antibody. Selected hybridomas are cloned to ensure monoclonality.
5. Production and purification:
• Cloned hybridoma cells are cultured to produce large quantities of the monoclonal antibody, which is then purified for use.

Vaccine formulations

Vaccine formulations are designed to induce an immune response and protect against specific diseases. They can include live attenuated vaccines, which contain weakened pathogens; inactivated vaccines, containing killed pathogens; subunit, recombinant, polysaccharide and conjugate vaccines, which use specific parts of the pathogen; toxoid vaccines, which use inactivated toxins; mRNA vaccines, which use messenger RNA to instruct cells to produce a pathogen protein; and viral vector vaccines, which use a modified virus to deliver genetic material. Components of vaccines typically include antigens to stimulate the immune response, adjuvants to enhance this response, stabilizers to maintain vaccine potency and preservatives to prevent contamination.

Vaccine formulations are carefully designed to induce an immune response that provides protection against specific diseases. These formulations include various types of vaccines, each with unique characteristics and components to ensure efficacy, stability and safety.

Live attenuated vaccines contain live pathogens that have been weakened so they cannot cause disease in healthy individuals. Examples include the measles, mumps, rubella (MMR) vaccine and the varicella (chickenpox) vaccine. These vaccines usually elicit a strong, long-lasting immune response but are not suitable for immunocompromised individuals due to the potential risk of reversion to a virulent form.

Inactivated (killed) vaccines contain pathogens that have been killed or inactivated, preventing them from replicating. Examples include the inactivated polio vaccine (IPV) and the hepatitis A vaccine. These vaccines are safe for immunocompromised individuals but often induce a weaker immune response compared to live vaccines, necessitating booster shots to maintain immunity.

Subunit, recombinant, polysaccharide and conjugate vaccines contain only specific parts of the pathogen, such as proteins or sugars, that best stimulate the immune system. Examples include the hepatitis B vaccine and the human papillomavirus (HPV) vaccine. These vaccines typically have a lower risk of side effects and focus the immune response on critical parts of the pathogen, though they may require adjuvants and multiple doses to be fully effective.

Toxoid vaccines contain inactivated toxins produced by the pathogen. Examples include the diphtheria and tetanus vaccines. These vaccines target the harmful toxins rather than the pathogen itself, providing immunity against toxin-mediated diseases. However, they may require periodic boosters to maintain long-term immunity.

mRNA vaccines use messenger RNA to instruct cells to produce a protein from the pathogen, which triggers an immune response. Examples include the Pfizer-BioNTech and Moderna COVID-19 vaccines. This technology allows for rapid development and strong immune responses, though it requires ultra-cold storage and is relatively new, with long-term effects still being studied.

Viral vector vaccines use a modified virus (vector) to deliver genetic material from the pathogen to stimulate an immune response. Examples include the Johnson & Johnson and AstraZeneca COVID-19 vaccines. These vaccines can induce strong immune responses and are stable at refrigerator temperatures, but pre-existing immunity to the vector virus can potentially reduce their efficacy.

Components of vaccine formulations include antigens, which are the active components that stimulate an immune response. Adjuvants are substances added to enhance the immune response, making the vaccine more effective. Stabilizers are used to maintain the vaccine's potency during storage and transport, ensuring it remains effective until administration. Preservatives are included to prevent contamination and extend the vaccine's shelf life.

Understanding the different types of vaccine formulations and their components is crucial for developing effective immunization strategies and ensuring public health safety.

Conclusion

The formulation of biologics requires a multifaceted approach to overcome challenges related to stability and bioavailability. Advances in protein engineering, innovative delivery systems and alternative administration routes continue to drive improvements in this field. Future research and development will likely focus on further enhancing these strategies to ensure the successful deployment of biologics in therapeutic settings.

Acknowledgement

None.

Conflict of Interest

None.

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