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Chapter 9 Biopharmaceuticals Manufacturing CHE 714 Pharmaceutical Technology and Processes Dr. Ginette Turcotte From www.drugdevelopment-technology.com accessed Aug. 18, 2015 1 Course Objectives 1. Associate a few names of drugs with the names of their microbial or animal cell source(s) 2. Describe conditions to manufacture some biopharmaceuticals 3. Suggest potential manufacturing conditions (designing) for an (un)known biopharmaceutical, based on information about type of source, metabolism of a few candidate sources, and general bioreactor and purification principles 4. State specific Quality assurance and Validation steps for biopharmaceuticals 2 Outline How are biopharmaceuticals different from pharmaceuticals? How are they manufactured from microbial and animal cells? Cell metabolism and over-production Upstream Processing Bioreactors Downstream Processing Analyses and Scale-up Specific Quality Assurance and Validation - regulations: toxins, toxoids, pyrogens, biologic gmp, biosimilars, Canada’s Food and Drugs Act http://laws-lois.justice.gc.ca/eng/acts/F-27/index.html 3 What are Biopharmaceuticals? Biopharmaceuticals, also called therapeutics, biologics or biotherapeutics, are complex macromolecules (many of them are glycoproteins) that are grown from microbial, animal or plant cells, mostly through recombinant DNA technology for medicinal purposes. They all possess structural and biological activities that must be maintained throughout their production, their storage, their delivery, and their “life” inside the human body for adequate targeted effect. 4 Examples of biopharmaceuticals {5C22544A-7EE6-4342-B048-85BDC9FD1C3A}Commercial drug (r-protein1) Active Pharmaceutical Ingredient Manufacturer Potential source Dosage form Humatrope; Serostim Human growth hormone (rHGH) Lilly; Serono E. coli Injection Humulin; Novolin Human insulin (BHI) Lilly; Novo Nordisk E. coli Injection Epogen Erythropoietin (EPO) Amgen Mammalian CHO cells Injection Filgrastim, sold as Neupogen; pegfilgrastim, sold as Neulasta Granulocyte colony-stimulating factor (G-CSF) Amgen E. coli Injection Aldurazyme alpha-L-iduronidase (rhIDU; laronidase) BioMarin Pharmaceutical and Genzyme Mammalian CHO cells Intravenous infusion Activase Tissue plasminogen activator (TPA) Genentech Mammalian CHO cells Injection Ceredase Glucocerebrosidase Genzyme Human placental tissue - withdrawn Intravenous infusion Avonex; Rebif Interferon (IF) beta-1a Biogen Idec; Serono Mammalian CHO cells Injection Betaseron Interferon (IF) beta-1b Schering E. coli Injection 1r-protein: recombinant protein 5 {5C22544A-7EE6-4342-B048-85BDC9FD1C3A}Commercial drug (r-viral) Active Pharmaceutical Ingredient Manufacturer Potential source Dosage form Avaxim Hepatitis A virus Inactivated (GBM strain) Sanofi Pasteur Diploid WI-38 human cell line Injection Engerix-B Envelope protein of the hepatitis B virus GlaxoSmithKline S. cerevisiae Injection Gardasil; Cervarix Human Papilloma Virus vaccine protein GlaxoSmithKline S. cerevisiae Injection Pneumo 23 Polysaccharides - meningitis Sanofi Pasteur S. pneumoniae Injection Varivax Live attenuated chicken pox virus Merck & Co., Inc. Diploid MRC5 cell line Injection Vaxigrip Inactivated influenza virus Sanofi Pasteur Eggs (chicken embryo) Injection 6 Biopharmaceuticals versus pharmaceuticals {5C22544A-7EE6-4342-B048-85BDC9FD1C3A}Comparison Pharmaceuticals Biopharmaceuticals Size Small molecules Complex macromolecules: 6 kDa (6,000 g/mol) - recombinant insulin 22 kDa - recombinant growth hormone > 280 kDa - recombinant factor VIII 1,000 kDa - recombinant von Willebrand factor Dosage form Solid, liquid Aqueous solutions Delivery means Pills; drops; creams; patches; etc. Muscular injections; intravenous infusions; blood transfusions; etc. Stability (half-life) Up to a few years – some aw problems Up to a few months – protein denaturation problems Bioavailability < 100% of the drug reaches site of action 100% when intravenously administered Etc.?! 7 Characteristics of biopharmaceuticals Protein structure/conformation – primary, secondary, tertiary, quaternary Glycosylation – neutral sugars and sialic acids covalently attached to specific amino acids of the protein, resulting in glycoprotein pH(I), pH at which a protein has no net charge – regulates solubility of protein in environment Colloidal properties due to size Aggregation – ex., insulin’s active form is a monomer Pharmacokinetics – faster-reacting drugs are usually smaller molecules (faster diffusion) 8 r-protein production Standard cloning protocol: Choice of host organism, commercial cloning vector (or plasmid) and gene transfer reagents (especially with animal cells) Preparation of cloning vector DNA – steps 3, 4 Preparation of DNA to be cloned – steps 2, 3, 4 Creation of recombinant DNA - steps 5, 6 Introduction of recombinant DNA into the host organism – step 7 Selection of organisms containing recombinant DNA – steps 8, 9 Screening for clones with desired DNA inserts and biological properties – step 11, etc. From Wikimedia Commons, accessed Aug. 18, 2015 9 Metabolic over-production of r-protein – host cells {5C22544A-7EE6-4342-B048-85BDC9FD1C3A}Microbial cells Animal cells Plant cells E. coli K12 and its derivatives U2OS cells, derived from human bone osteosarcoma Tobacco Agrobacterium HepG2 cells, derived from a human hepatocellular carcinoma Mays, corn Saccharomyces cerevisiae Human 293 cell line . Growth in cell suspension . High transfection yield with most gene transfer vehicles . Easily grown in suspension culture . Can be adapted to serum-free media Bacillus subtilis Baculovirus/insect Clostridium botulinum Chinese Hamster Ovary (CHO); mouse myeloma (NSO); baby hamster kidney (BHK); human embryo kidney (HEK-293); human retina-derived PER-C6 cells 10 Metabolic over-production of r-protein – vectors (plasmids) {5C22544A-7EE6-4342-B048-85BDC9FD1C3A}Microbial cells Animal cells Plant cells Plasmids of genetics variants of 293E and 293T cell lines . Highly active promoter, CMV1 in highly efficient expression cassette 1CMV: human cytomegalovirus Selection agent: second gene, applied a few days after gene transfer; on same plasmid, or not, as the biopharma-encoding gene Dihydrofolate reductase (DHFE), an enzyme involved in nucleotide metabolism Glutamine synthetase (GS) 11 Cationic polymer polyethylenimine (PEI) Linear and branched isoforms with a wide range of molecular weights and polydispersities – critical to efficient gene transfer activity 25 kDa Simple to use and effective for large-scale (multi-liters) suspensions of animal cells Minimal cytotoxic effects Works well in serum-free and in serum media Metabolic over-production of r-protein – Gene transfection vehicles 12 Production Process for a Biopharmaceutical 13 Upstream processing Downstream processing Schematic representation of the processes taking place inside a chromatographic column on the microscopic scale and the parameters commonly used to describe protein molecular properties, resin properties, and their interactions. Arrows indicate mass-transfer effects, solid arrows convection, dashed arrows diffusion, and the length of each arrow is qualitatively related to the magnitude of the effect. The exact definitions of each parameter may vary with the models applied and some models may require only a subset or even more than the presented parameters. Hanke, AT, Ottens M (2014). Purifying biopharmaceuticals: knowledge-based chromatographic process development. Trends in Biotechnology 32 (4): 210-220 doi:10.1016/j.tibtech.2014.02.001 14 Analyses FoR biopharmaceuticals Protein concentration – which reference method to use? Conformation, pI, glycosylation Activity, stability, purity Excipients, adjuvants, antioxidants, antimicrobials Contaminants (microbes; viral particles; pyrogen; endotoxins (LPS); DNA; proteins) 15 Scale-up Batch to continuous; fed-batch or perfusion mode; immobilized cells or not; adherent to suspended mammalian cell cultures? T; O2 transfer; heat load and transfer; surface area to volume ratio; cell viability Embryonic egg-based cultures? Systems biology with genomics, proteomics, metabolomics and bioinformatics How to scale-up upstream as well as downstream processes? 16 Specific Quality Assurance and Validation for Biopharmaceuticals {5C22544A-7EE6-4342-B048-85BDC9FD1C3A}Validation Pharmaceuticals Biopharmaceuticals Moist heat sterilization cycles NA Spores of Bacillus stearothermophilus; D-value Pyrogens; endotoxins ? ? Classical fermentation NA Low MW products (antibiotics, amino acids, vitamins, and carbohydrates) Cell banking NA ? Frequency of monitoring ? ? but may be more than with pharmaceuticals Aseptic manufacturing ? ? but may be more than with pharmaceuticals Cold chain ? Cold storage; freeze-thaw cycles 17 Regulatory evaluation of Biosimilars Three characteristics to be met by a Biosimilar: Post-translational modifications Three-dimensional structures Protein aggregation See next slide 18 a As mass increases in peptides, the sugar fucose in the r-protein, was found in another location from that of the original protein; technique is tandem mass spectrometry (MS-MS) b Most glycosylation changes in the protein conformation of immunoglobulin G (IgG) were seen in the region 292-308 of amino acids by hydrogen-deuterium exchange MS (HDX-MS) c The aggregation of insulin (dimer versus monomer) can be detected using ion-mobility spectrometry From Berkovitz SA, Engen JR, Mazzeo JR, Jones GB (2012) Analytical tools for characterizing biopharmaceuticals and the implications for biosmilars. Nature Reviews Drug Discovery 11, 527-540, doi:10.1038/nrd3746 19 Conclusion For a process engineer, a biopharmaceutical manufacturing plant substantially differs from a pharmaceutical manufacturing plant: At some point, live cells are used that may or may not be dangerous for people because of their potentially unknown nature No material (air, contaminants, etc.) from within is allowed outside the manufacturing plant We will study each aspects of cell metabolism and over-production, upstream and downstream processing (including bioreactors and scale-up), required analyses on products, plus specific quality assurance and validation requirements 20
