©1999 Timothy G. Standish Pharmaceutical Biotechnology PHG 424 Mounir M. Salem, Ph.D. King Saud University College of Pharmacy Departments of Pharmaceutics/ Pharmacognosy

©1999 Timothy G. Standish Biotechnology Products

©1999 Timothy G. Standish Microbiological Consideration  Most proteins are administered parenterally and have to be sterile.  In general proteins are sensitive to heat and other regularly used sterilization methods; they can’t withstand autoclaving, gas sterilization, or sterilization by ionizing radiation. Consequently, sterilization of the end product is not possible.  Therefore, protein pharmaceuticals have to be assembled under aseptic conditions.

©1999 Timothy G. Standish Microbiological Consideration…cont.  Equipment and excipients are treated separately and autoclaved, or sterilized by dry heat (>160 ºC), chemical treatment or radiation to minimize bioburden.  As a recombinant DNA products are grown in microorganisms, these should be tested for viral contamination and appropriate measures should be taken if viral contaminations occur.  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 processes should be minimized.

©1999 Timothy G. Standish  Bioburden or microbial limit testing is performed on pharmaceutical products and medical products as a quality control measure. Products or components used in the pharmaceutical or medical field require control of microbial levels during processing and handling.  Bioburden of raw material as well as finished pharmaceutical products can help to determine whether the product complies with the requirements of the BP, Eur. or USP.  Bioburden is the number of microorganisms with which an object is contaminated. This unit is measured in CFU per gram of product. Microbiological Consideration…cont.

©1999 Timothy G. Standish Excipients used in biotechnology products Introduction: Active ingredient. Solubility enhancers. Anti-adsorption and anti-aggregation agents. Buffer components. Preservatives and anti-oxidants. Osmotic agents. Carrier system.

©1999 Timothy G. Standish Excipients used in biotechnology products Solubility Enhancers: In general, proteins may have a tendency to aggregate and precipitate. Different methods can be used to enhance solubility, including: selection of proper pH and ionic strength conditions, addition of amino acid or surfactants. Selection of appropriate enhancers is mainly dependent on: type of protein involved and mechanism of action of the enhancer.

©1999 Timothy G. Standish Anti-adsorption and anti-aggregation agents: Anti-adsorption agents are added to reduce adsorption of the active protein to interfaces. Albumin has a strong tendency to adsorb to surfaces and therefore added in relatively high concentrations to protein formulation as an anti-adhesion agent. Excipients used in biotechnology products

©1999 Timothy G. Standish 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. Even short, temporary pH changes can cause aggregation. These conditions can occur, for example, during the freezing step in the freeze-drying process. Excipients used in biotechnology products

©1999 Timothy G. Standish Excipients used in biotechnology products Preservative and Anti-oxidants: Methionine, cysteine, tryptophan, tyrosine and histidine are amino acids that are readily oxidized (oxidative degradation). Replacement of oxygen by inert gases in the vials helps to reduce oxidative stress. Moreover, the addition of anti-oxidants such as ascorbic acid or sodium formaldehyde sulfoxylate. Certain proteins are formulated in containers designed for multiple injection schemes. Preservatives are usually added to minimize growth of microorganisms and thus reduce chance for contamination.

©1999 Timothy G. Standish Therapeutic Proteins Insulin (diabetes) Interferon  (relapsing MS) Interferon  (granulomatous) TPA (heart attack) TPA: Tissue plasminogen activator

©1999 Timothy G. Standish Actimmune (If  ) Activase (TPA) BeneFix (F IX) Betaseron (If  ) Humulin Novolin Pegademase (AD) Epogen Regranex (PDGF) Novoseven (F VIIa) Intron-A Neupogen Pulmozyme Infergen Therapeutic Proteins…

©1999 Timothy G. Standish The Problem with Proteins Very large and unstable molecules Structure is held together by weak noncovalent forces Easily destroyed by relatively mild storage conditions Easily destroyed/eliminated by the body Hard to obtain in large quantities Therapeutic Proteins…

©1999 Timothy G. Standish The Problem with Proteins (in vivo) Elimination by B and T cells Proteolysis by endo/exo peptidases Small proteins (<30 kD) filtered out by the kidneys very quickly Unwanted allergic reactions may develop (even toxicity) Loss due to insolubility/adsorption Therapeutic Proteins…

©1999 Timothy G. Standish

The Problem with Proteins (in vitro) Denaturation Aggregation Precipitation Adsorption Deamidation Oxidation Disulfide exchange Proteolysis Noncovalent Covalent Therapeutic Proteins…

©1999 Timothy G. Standish Noncovalent Processes Denaturation Adsorption Therapeutic Proteins…

©1999 Timothy G. Standish Aggregation Precipitation Therapeutic Proteins… Noncovalent Processes

©1999 Timothy G. Standish Covalent Processes Deamidation - conversion of Asn-Gly sequences to  -Asp-Gly or  -Asp-Gly Oxidation - conversion RSR’ to RSOR’, RSO 2 R’ or RSO 3 R’ (Met & Cys) Disulfide exchange - RS - + R’S-SR’’ goes to RS-SR’’ + R’S - (Cys) Proteolysis - Asp-Pro, Trypsin (at Lys) or Chymotrypsin (at Phe/Tyr) Therapeutic Proteins…

©1999 Timothy G. Standish Deamidation Therapeutic Proteins…

©1999 Timothy G. Standish How to Deal with These Problems? Storage Formulation Delivery Pharmaceutics Therapeutic Proteins…

©1999 Timothy G. Standish Storage - Refrigeration Low temperature reduces microbial growth and metabolism Low temperature reduces thermal or spontaneous denaturation Low temperature reduces adsorption Freezing is best for long-term storage Freeze/Thaw can denature proteins Therapeutic Proteins…

©1999 Timothy G. Standish Storage - Packaging Smooth glass walls best to reduce adsorption or precipitation Avoid polystyrene or containers with silanyl or plasticizer coatings Dark, opaque walls reduce oxidation Air-tight containers or argon atmosphere reduces air oxidation Therapeutic Proteins…

©1999 Timothy G. Standish Storage - Additives Addition of stabilizing salts or ions (Zn2+ for insulin) Addition of polyols (glycerol and/or polyethylene glycol) to solubilize Addition of sugars or dextran to displace water or reduce microbe growth Use of surfactants (CHAPS) to reduce adsorption and aggregation Therapeutic Proteins…

©1999 Timothy G. Standish Storage - Freeze Drying Only cost-effective means to prepare solid, chemically active protein Best for long term storage Removes a considerable amount of water from protein lattice, so much so, that some proteins are actually deactivated Therapeutic Proteins…

©1999 Timothy G. Standish Shelf Life of Protein Protein can be stored: as an aqueous solution, in freeze dried form, or in dried form in a compacted state (tablet). Stability of protein solutions strongly depends on factors such as pH, ionic strength, temperature and the presence of stabilizers.

©1999 Timothy G. Standish Shelf Life of Protein Freeze-drying of Proteins: The abundant presence of large amount of water in he proteins in solution makes it difficult to maintain preferred self life (i.e. 2 years) for protein products. Freeze drying may provide a good stability because of the water removal through sublimation and not by evaporation. Freezing step, primary drying, secondary drying are the major three steps in freeze drying process.

©1999 Timothy G. Standish Freeze Drying Freeze liquid sample in container Place under strong vacuum Solvent sublimates leaving only solid or nonvolatile compounds Reduces moisture content to <0.1% Therapeutic Proteins…

©1999 Timothy G. Standish Protein Pharmaceutics Storage Formulation Delivery

©1999 Timothy G. Standish The Problem with Proteins (in vivo) Elimination by B and T cells Proteolysis by endo/exo peptidases Small proteins (<30 kD) filtered out by the kidneys very quickly Unwanted allergic reactions may develop (even toxicity) Loss due to insolubility/adsorption Therapeutic Proteins…

©1999 Timothy G. Standish Protein Formulation Protein sequence modification (site directed mutagenisis) PEGylation Proteinylation Microsphere/Nanosphere encapsulation Formulating with permeabilizers Therapeutic Proteins…

©1999 Timothy G. Standish Site Directed Mutagenesis E343H Therapeutic Proteins…

©1999 Timothy G. Standish Site Directed Mutagenesis Allows amino acid substitutions at specific sites in a protein will reduce likelihood of oxidation Strategic placement of cysteines to produce disulfides to increase Tm Protein engineering (size, shape, etc.) Therapeutic Proteins…

©1999 Timothy G. Standish PEGylation + O O O O Therapeutic Proteins…

©1999 Timothy G. Standish PEGylation PEG is a non-toxic, hydrophilic, FDA approved, uncharged polymer Increases in vivo half life (4-400X) Decreases immunogenicity Increases protease resistance Increases solubility & stability Reduces depot loss at injection sites Therapeutic Proteins…

©1999 Timothy G. Standish Proteinylation + Protein Drug ScFv (antibody) Therapeutic Proteins…

©1999 Timothy G. Standish Attachment of additional or secondary (nonimmunogenic) proteins for in vivo protection Increases in vivo half life (10X) Cross-linking with Serum Albumin Cross-linking or connecting by protein engineering with antibody fragments Therapeutic Proteins… Proteinylation

©1999 Timothy G. Standish Microsphere Encapsulation 100  m Therapeutic Proteins…

©1999 Timothy G. Standish Encapsulation Process involves encapsulating protein or peptide drugs in small porous particles for protection from “insults” and for sustained release Two types of microspheres –nonbiodegradable –biodegradable Therapeutic Proteins…

©1999 Timothy G. Standish Types of Microspheres Nonbiodegradable –ceramic particles –polyethylene co-vinyl acetate –polymethacrylic acid/PEG Biodegradable (preferred) –gelatin –polylactic-co-glycolic acid (PLGA) Therapeutic Proteins…

©1999 Timothy G. Standish PLGA - Structure Therapeutic Proteins…

©1999 Timothy G. Standish Microsphere Release Hydrophilic (i.e. gelatin) –best for burst release Hydrophobic (i.e. PLGA) –good sustained release (esp. vaccines) –tends to denature proteins Hybrid (amphipathic) –good sustained release –keeps proteins native/active Therapeutic Proteins…

©1999 Timothy G. Standish Release Mechanisms Therapeutic Proteins…

©1999 Timothy G. Standish Peptide Micelles Therapeutic Proteins…

©1999 Timothy G. Standish Peptide Micelles Small, viral sized (10-50 nm) particles Similar to lipid micelles Composed of peptide core (hydrophobic part) and PEG shell (hydrophilic part) Peptide core composition allows peptide/protein solubilization Also good for small molecules Therapeutic Proteins…

©1999 Timothy G. Standish Peptide Synthesis Therapeutic Proteins…

©1999 Timothy G. Standish Peptide-PEG monomers PeptidePEG Hydrophobic block Hydrophilic block CH 2 -CH 2 -O-CH 2 -CH 2 -O-CH 2 -CH 2 -O-CH Therapeutic Proteins…

©1999 Timothy G. Standish Peptide Micelles Therapeutic Proteins…

©1999 Timothy G. Standish Targeted Micelles Therapeutic Proteins…

©1999 Timothy G. Standish Nanoparticles for Vaccine Delivery to Dendritic Cells Dendritic Cells -‘sentries’ of the body Eat pathogens and present their antigens to T cells Secret cytokines to direct immune responses Therapeutic Proteins…

©1999 Timothy G. Standish Nanoparticles for Vaccine Delivery Mimic pathogen surface characteristics Antigen for controlled delivery within Dendritic Cells Selective activation of cytokine genes in Dendritic Cells Applications in Therapeutic Vaccines (e.g., cancer, AIDS, HBV, HCV) Therapeutic Proteins…

©1999 Timothy G. Standish Polymeric Nanoparticle Uptake by Human DCs: Confocal Image Therapeutic Proteins…

©1999 Timothy G. Standish Permeabilizers (Adjuvants) Salicylates (aspirin) Fatty acids Metal chelators (EDTA) Anything that is known to “punch holes” into the intestine or lumen Therapeutic Proteins…

©1999 Timothy G. Standish Protein Formulation (Summary) Protein sequence modification (site directed mutagenisis) PEGylation Proteinylation Microsphere/Nanosphere encapsulation Formulating with permeabilizers Therapeutic Proteins…

©1999 Timothy G. Standish Protein Pharmaceutics StorageFormulation Delivery

©1999 Timothy G. Standish

Routes of Delivery Parenteral (injection) Oral or nasal delivery Patch or transdermal route Other routes –Pulmonary –Rectal/Vaginal –Ocular

©1999 Timothy G. Standish Parenteral Delivery Intravenous Intramuscular Subcutaneous Intradermal

©1999 Timothy G. Standish Parenteral Delivery Route of delivery for 95% of proteins Allows rapid and complete absorption Allows smaller dose size (less waste) Avoids first pass metabolism Avoids protein “unfriendly zones” Problems with overdosing, necrosis Local tissue reactions/hypersensitivity Everyone hates getting a needle

©1999 Timothy G. Standish Exubera (Inhaled Insulin) Exubera, a dry-powder form of insulin, is inhaled with a special device similar to an asthma inhaler Exubera normalized blood sugar levels as well as injections did Patients taking inhaled insulin also reported greater satisfaction and quality of life (for 18+ only) About 1/5 study subjects developed a mild cough with inhaled insulin Product pulled in Oct Pfizer

©1999 Timothy G. Standish Oral Insulin (Oralin)

©1999 Timothy G. Standish Oral Insulin (Oralin/Oral-lyn) Bucchal aerosol delivery system developed by Generex (Approved in Ecaudor and India) Insulin is absorbed through thin tissue layers in mouth and throat Insulin is formulated with a variety of additives and stabilizers to prevent denaturation on aerosolization and to stabilize aerosol particles

©1999 Timothy G. Standish BioSante’s BioOral Insulin The BioOral formulation was developed by aggregating caseins (the principle protein in milk) around a proprietary formulation of CAP (calcium phosphate nanoparticle), polyethylene glycol (PEG, a polymer) and insulin by scientists at BioSante's research center

©1999 Timothy G. Standish Oral Delivery by Microsphere pH 2 pH 7

©1999 Timothy G. Standish pH Sensitive Microspheres Gel/Microsphere system with polymethacrylic acid + PEG In stomach (pH 2) pores in the polymer shrink and prevent protein release In neutral pH (found in small intestine) the pores swell and release protein Process of shrinking and swelling is called complexation (smart materials)

©1999 Timothy G. Standish Patch Delivery

©1999 Timothy G. Standish Mucoadhesive Patch Adheres to specific region of GI tract Ethylcellulose film protects drugs from proteolytic degradation Composed of 4 layers –Ethylcellulose backing –Drug container (cellulose, citric acid) –Mucoadhesive glue (polyacrylic acid/PEG) –pH Surface layer (HP-55/Eudragit)

©1999 Timothy G. Standish Patch Delivery

©1999 Timothy G. Standish GI-MAPS Layers pH sensitive surface layer determines the adhesive site in the GI tract Gel-forming mucoadhesive layer adheres to GI mucosa and permits controlled release - may also contain adjuvants Drug containing layer holds powders, dispersions, liquids, gels, microspheres, Backing layer prevents attack from proteases and prevents luminal dispersion

©1999 Timothy G. Standish Transdermal Patches

©1999 Timothy G. Standish Transdermal Patches Proteins imbedded in a simple matrix with appropriate additives Patch is coated with small needles that penetrate the dermal layer Proteins diffuse directly into the blood stream via capillaries Less painful form of parenteral drug delivery

©1999 Timothy G. Standish Close-up of Patch Pins

©1999 Timothy G. Standish Biocapsules

Summary Protein pharmaceuticals are (and will be) the most rapidly growing sector in the pharmaceutical repertoire Most “cures” for difficult diseases (Alzheimers, cancer, MS, auto-immune diseases, etc.) will probably be found through protein drugs

©1999 Timothy G. Standish Summary BUT Proteins are difficult to work with Most protein delivery is via injection Newer methods are appearing Oral delivery using “smart materials” is looking promising Over the coming 3-4 years more protein drugs will have oral formulations

©1999 Timothy G. Standish