Formulation of Biotech Products I (Biopharmaceutical Considerations) Lecture 2 PDF

Formulation of Biotech Products I (Biopharmaceutical Considerations) Lecture 2 Assist. Prof. Dr. Mohammed Talib PhD in Biotechnology College of Pharmacy University of Misan Microbial considerations 1- Sterility 2- Vi...

Formulation of Biotech Products I (Biopharmaceutical Considerations) Lecture 2 Assist. Prof. Dr. Mohammed Talib PhD in Biotechnology College of Pharmacy University of Misan Microbial considerations 1- Sterility 2- Viral decontamination 3- Pyrogen removal 2 Sterility ❖ Most proteins are administered parenterally and have to be sterile. ❖ They cannot withstand autoclaving, gas sterilization, or sterilization by ionizing radiation. For this reason : ❑ Protein pharmaceuticals have to be assembled under aseptic conditions. 3  Microbiological Considerations Sterility: ✓Filtration techniques are used for removal of micro- bacterial contaminants. (E.g.: Recombinant/purified protein vaccines consist of protein antigens like Hepatitis B vaccine I.M.) ✓The final “sterilizing” step before filling the vials is filtration through 0.2 or 0.22 µm membrane filters. ✓Assembly of the product is done in rooms with laminar airflow that is filtered through high efficiency particulate air (HEPA) filters. ✓Well-trained technicians / or operators wearing protective cloths (face masks, hats, gloves, or head- to-toe overall garments) should operate the facility. 4  Microbiological Considerations Viral Decontamination ❖ As recombinant DNA products are grown in microorganisms, these organisms should be tested for viral contaminants. ❖Appropriate measures should be taken if viral contamination occurs ❖ Example: Excipients with a certain risk factor such as blood-derived human serum albumin should be carefully tested before use and their presence in the formulation process should be minimized. 5 Viral Decontamination Challenges 1- Preservation of Protein Function: Harsh treatments (e.g., high heat or strong chemicals) can denature proteins or reduce their activity. 2- Effectiveness Across Virus Types: Some viruses, especially non-enveloped ones, are more resistant to certain decontamination methods. 3- Scalability: Techniques need to be efficient for both small- scale research and large-scale manufacturing. 6  Microbiological Considerations Pyrogen Removal: “ Pyrogens are fever-inducing substances usually derived from microorganisms [endotoxins or lipopolysaccharide (LPS)] and when present systemically in sufficient quantity can lead to severe signs of inflammation, shock, multiorgan failure, and sometimes even death in humans”. Source:P.E. Boucher, in Immunopotentiators in Modern Vaccines (Second Edition), 2017. Equipment and container are treated at temperatures above 160 C for prolonged periods (e.g., 30 minutes dry heat at 250 C) to minimize the bio-burden. 7 General Structure of Pyrogens/or Endotoxins / or LPS 8 Ref: Franco et al.,2018 https://doi.org/10.3390/toxins10080331 9 Microbiological Considerations…continued Pyrogen Removal from proteins: ❖ Ion exchange chromatographic procedures (utilizing its negative charge) can effectively reduce endotoxin levels in solution. ❖ Activated charcoal or other materials with large surfaces offering hydrophobic interactions to remove of endotoxins immediately before filling the final container. The principle of Ion Exchange Chromatography. ❖ Oxidation: Endotoxins can also be The separation occurs by reversible exchange of ions inactivated by oxidation (e.g., peroxide). between the ions present in the solution and those present in the ion exchange resin.. 10  ❖ Pyrogen Removal by Specific Kits For example: BcMag™ Quick Endotoxin Removal Kit / 386 $ for 2ml Uses a magnetic microsphere covalently immobilized with a high density of polymyxin B to remove endotoxin. It is specially designed for quick endotoxin removal from various sample types. Polymyxin B, a peptide antibiotic, has a very high binding affinity for the lipid A moiety of most endotoxins. 11 Excipients Used in Parenteral Formulation The nature of the protein (e.g., lability) and its therapeutic use (e. g., multiple injection systems) can make these formulations quite complex in terms of: ✓Excipient profile ✓Technology (freeze-drying, aseptic preparation). 12  Excipients Used in Parenteral Formulation Components can be found in the presently marketed formulations: – Active ingredient – Solubility enhancers – Anti-adsorption and anti-aggregation agents – Buffer components – Preservatives and antioxidants – Osmotic agents – Lyoprotectants – Carrier system. 13  Excipients Used in Parenteral Formulation Solubility Enhancers: – Proteins may tend to aggregate and precipitate, and approaches that can be used to enhance solubility include: Selection of the proper pH and ionic strength conditions. Addition of amino acids such as lysine or arginine which used to solubilize tissue plasminogen activator (t-PA). Surfactants such as sodium dodecylsulfate. 14 Excipients Used in Parenteral Formulation Anti-Adsorption and Anti-Aggregation Agents: – Anti-adsorption agents are added to reduce adsorption of the active protein to interfaces. – These interfaces can be water/air, water/container wall or interfaces formed between the aqueous phase and materials used to administrate the drug (e.g., catheter, needle). 15 Excipients Used in Parenteral Formulation As an example, the proposed mechanism for aggregation of insulin in aqueous media through contact with a Reversible self-association of insulin, its adsorption to the hydrophobic interface and irreversible aggregation hydrophobic surface (or water– in adsorbed protein films. Each circle represents a monomeric insulin molecule. air interface). Source: Adapted from Thurow and Geisen 1984 16 Excipients Used in Parenteral Formulation Anti-Adsorption and Anti-Aggregation Agents: – Albumin strongly tends to adsorb to surfaces and compete with therapeutics protein and is therefore added in relatively high concentrations (E.g., 1%). – Surfactants molecules readily adsorb to hydrophobic interfaces with their own hydrophobic groups and render this interface hydrophilic by exposing their hydrophilic groups to the aqueous phase. 17 Excipients Used in Parenteral Formulation Buffer Components: – Buffer selection is an important part of the formulation process, because of the pH dependence of protein solubility and physical, and chemical stability. – Buffer systems regularly encountered in biotech formulations are phosphate, citrate, and acetate. – Temporary pH changes can cause aggregation. 18 Excipients Used in Parenteral Formulation Antioxidants: – Methionine, cysteine, tryptophan, tyrosine, and histidine are amino acids that are readily oxidized. – Proteins rich in these amino acids are liable to oxidative degradation. ✓Replacement of oxygen by inert gases in the vials helps to reduce oxidative stress. ✓The addition of antioxidants such as ascorbic acid or acetylcysteine can be considered. 19 Excipients Used in Parenteral Formulation Preservatives : – Certain proteins are formulated in containers designed for multiple injection schemes. – After administering the first dose, contamination with microorganisms may occur and preservatives are needed to minimize growth. – These preservatives are present in concentrations that are bacteriostatic rather than bactericidal in nature. – Antimicrobial agents are the mercury-containing phenyl mercuric nitrate and p-hydroxybenzoic acids, phenol, benzyl alcohol and chlorobutanol 20 Excipients Used in Parenteral Formulation Osmotic agents: – Adjusting the tonicity of parenteral protein products is an integral part of the preparation. – Saline and mono- or disaccharide solutions are commonly used. (These excipients may not be inert; they may influence protein structural stability). – Sugars and polyhydric alcohols can stabilize the protein structure through the principle of “preferential exclusion” 21 Excipients Used in Parenteral Formulation Osmotic Agents: – Preferential exclusion These additives (water structure promoters) enhance the interaction of the solvent with the protein and are themselves excluded from the protein surface layer; the protein is preferentially hydrated. Unfortunately, a strong “preferential exclusion” effect enhances the tendency of proteins to self-associate. 22 Shelf Life of Protein-Based Pharmaceuticals Proteins can be stored as:- –An aqueous solution. –In freeze-dried form. –In dried form in a compacted state (tablet). 23 Freeze Drying of Proteins Proteins in solution often do not meet the preferred stability requirements for industrially produced pharmaceutical products (> 2 years), even when kept permanently under refrigerator conditions. During freeze-drying, water is removed through sublimation and not by evaporation. Freeze drying of a protein solution without the proper excipients causes irreversible damage to the protein. 24 Freeze Drying of Proteins Excipients: – Mannitol – Glycine – Sugar – Albumin – Dextran 25 Freeze Drying of Proteins Three stages in the freeze-drying process of protein formulations: 1. Freezing: In the freezing step the temperature of the aqueous system in the vials is lowered. 2. Primary Drying: In the primary drying stage sublimation of the water mass in the vial is initiated by lowering the pressure. 3. Secondary drying: Removal of water interacting with the protein and excipients. The temperature then rises gradually, e.g., from – 40 C to 20 C. 26  Delivery of Proteins: Routes of Administration and Absorption Enhancement The Parenteral Route of Administration: It defined as administration via those routes where a needle is used including Intravenous (IV), intramuscular (IM), subcutaneous (SC) and intraperitoneal (IP) injections. – It suffices here to state that the blood half-life of biotech products can vary over a wide range. E.g : the circulation half-life of t-PA is a few minutes, while monoclonal antibodies (MAb) reportedly have half life for a few days. 27 Delivery of Proteins: Routes of Administration and Absorption Enhancement The Parenteral Route of Administration: – A simple way to expand the mean residence time for short half-life proteins is to switch from IV to IM or SC administration. – Why can this change therapeutic effects of protein products??? 28 Delivery of Proteins: Routes of Administration and Absorption Enhancement The Parenteral Route of Administration: – These changes are related to: The prolonged residence time at the IM or SC site of injection The enhanced exposure to degradation reactions (such as peptidases) Differences in disposition. 29 Routes uptake of SC or IM injected drug / Differences in Disposition Source Adapted from Supersaxo et al., 1990 30 Delivery of Proteins: Routes of Administration and Absorption Enhancement The Oral Route of Administration: – Oral delivery of protein drugs would be preferable, because it is patient friendly and no intervention by a healthcare professional is necessary to administer the drug. – Oral bioavailability, however, is usually very low. Why???? 31 Delivery of Proteins: Routes of Administration and Absorption Enhancement The Oral Route of Administration: – The two main reasons for this failure of uptake are: 1- Protein degradation in the gastrointestinal (GI) tract a 2- Poor permeability of the wall of the GI tract in case of a passive transport process. – Nevertheless, some vaccines are used orally ! 32 Delivery of Proteins: Routes of Administration and Absorption Enhancement The Oral Route of Administration: – For oral vaccines the above-mentioned hurdles of degradation and permeation are not necessarily prohibitive. – For oral immunization, only a (small) fraction of the antigen (protein) has to reach its target site to elicit an immune response. – The target cells are lymphocytes and antigen presenting accessory cells located in Peyer’s patches. 33  Alternative Routes of Administration….continued In January 2006, the US FDA approved the first formulation of inhaled insulin (Exubera®, Pfizer Labs, New York, NY) for clinical use in nonsmoking adults with type 1 or type 2 diabetes without pulmonary disease. ❖ Pulmonary inhalation of insulin is specifically tested for mealtime glucose control. ❖ Uptake of insulin is faster than after a regular SC insulin injection (peak 5–60 minutes versus 60–180 minutes). ❖ Thus, Inhalation technology plays a critical role when considering the prospects of the pulmonary route for the systemic delivery of therapeutic proteins 34 The fraction of insulin ultimately absorbed depends on:- 1. Route of Administration: Subcutaneous, intravenous, inhalation, or experimental routes like oral or transdermal. 2. Injection Site: Absorption varies by location (abdomen > arms > thighs/buttocks). 3. Blood Flow: Increased blood flow (e.g., exercise, heat) enhances absorption, while reduced flow slows it. 4. Insulin Formulation: Rapid-, intermediate-, or long-acting insulins differ in absorption rates. 5. Dose and Concentration: Larger doses and higher concentrations can slow absorption. 6. Tissue Factors: Fat thickness, lipohypertrophy, and scar tissue impact absorption efficiency. 7. Patient-Specific Factors: Circulatory health, metabolic rate, and hydration levels affect absorption. 8. Temperature and Physical Activity: Warmer temperatures and activity near the injection site improve absorption. 35  Alternative Routes of Administration to the oral route for Biopharmaceuticals Nasal Advantages: It is easily accessible, fast uptake, and has a proven track record with several “conventional” drugs. It probably has lower proteolytic activity than in the Gl tract, and it avoids the first-pass effect. Spatial containment of absorption enhancers is possible. Disadvantages: Reproducibility (in particular under pathological conditions), safety (e.g., ciliary movement), low bioavailability for proteins. Pulmonary Advantages relatively easy to access, fast uptake, proven track record with “conventional” drugs, substantial fractions of insulin are absorbed, lower proteolytic activity than in the Gl tract, avoidance of hepatic first-pass effect, spatial containment of absorption enhancers is possible. Disadvantage reproducibility (in particular under pathological conditions, smokers/nonsmokers), safety (e.g., immunogenicity), presence of macrophages in the lung with high affinity for particulates. 36 Rectal Advantages easily accessible, partial avoidance of hepatic first-pass, probably lower proteolytic activity than in the upper parts of the Gl tract, spatial containment of absorption enhancers is possible, proven track record with a number of “conventional” drugs. Disadvantages low bioavailability for proteins. Buccal Advantages easily accessible, avoidance of hepatic first pass, probably lower proteolytic activity than in the lower parts of the Gl tract, spatial containment of absorption enhancers is possible. Disadvantages low bioavailability of proteins. 37 Transdermal Advantages: easily accessible, avoidance of hepatic first-pass effect, removal of formulation if necessary is possible, spatial containment of absorption enhancers, proven track record with “conventional” drugs, sustained/controlled release possible. Disadvantages: low bioavailability of proteins. 38  Approaches to Enhance Bioavailability of Proteins 1) Increase the permeability of the absorption barrier via: - Addition of fatty acids/phospholipids, bile salts, phenylglycine, ester, and ether type (non)-ionic detergents, salicylate derivatives, derivatives of fusidic acid. - Iontophoresis: a transdermal electrical current is induced by positioning two electrodes on different places on the skin. 2) Decrease peptidase activity at the site of absorption and along the “absorption route”: aprotinin, bacitracin, boroleucin, borovaline. 3) Enhance resistance against degradation by modification of the molecular structure. 4) Prolongation of exposure time (e.g., bio-adhesion technologies). 39 Many thanks 40