PMB 501 Pharmaceutical Biotechnology II Lecture Slides PDF

10/19/2023 AFE BABALOLA UNIVERSITY, ADO EKITI LECTURE OUTLINE: COLLEGE OF PHARMACY Cell lines, cultures and cell propagation. DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY AND BIOTECHNOLOGY Cell, tissue and organ culturing. Factor...

10/19/2023 AFE BABALOLA UNIVERSITY, ADO EKITI LECTURE OUTLINE: COLLEGE OF PHARMACY Cell lines, cultures and cell propagation. DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY AND BIOTECHNOLOGY Cell, tissue and organ culturing. Factors affecting cell culturing. COURSE CODE: PMB 501 Sources of Biopharmaceuticals. LECTURE TITLE: PHARMACEUTICAL Production of biopharmaceuticals, quality control and BIOTECHNOLOGY II validation. Biosafety-classification and levels. Biohazards and LECTURER: DR O. S. ALABI containment. 1 2 CELL LINES, CULTURES AND CELL PROPAGATION Cell culture technology has successfully integrated various disciplines, including cell biology, genetic engineering, protein chemistry, genomics, and chemical engineering, and is now the established method of CELL LINES, CULTURES AND CELL producing a number of important proteins. PROPAGATION Cell culture technology–derived products are currently used as medicines to prevent and treat serious diseases such as cancer, viral infections, heredity deficiencies, and a variety of chronic diseases. The products are proven to be safe, effective, and economical. 3 4 1  10/19/2023  Cell culture Most cells require a surface or an artificial substrate Cell culture: process by which cells of interest are isolated (adherent or monolayer culture) whereas others can be from living tissue of animals or plants and subsequently grown free floating in liquid culture medium (suspension grown under controlled conditions, generally outside their culture). natural (artificial) environment (in vitro). The physico-chemical properties of the environment such as The cells may be removed from the tissue directly and the culture medium’s pH, osmotic pressure, temperature, disaggregated by enzymatic or mechanical means before are also regulated appropriately depending on the cell type. cultivation, or they may be derived from a cell strain that has already been established (cell line ). The lifespan of most cells is genetically determined, but some cells can be “transformed” into immortal cells (cell The controlled conditions vary for each cell type, but lines) which will reproduce indefinitely if the optimal generally consist of a suitable vessel with a substrate or conditions are provided and maintained. medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, The stages in cell culture include: Primary and secondary hormones, and gases (CO2, O2). (Cell line) cultures. 5 6 Primary cell cultures Primary cell cultures are the most representative of normal A stage of culture where tissue is isolated from an organism, tissues, and are still valuable in some areas of virology, disaggregated into cells and proliferated in a suitable particularly for virus isolation. medium under appropriate conditions such as temperature, However, they are labour-intensive to obtain, and are pH, osmotic pressure etc is termed Primary culture. heterogeneous in nature. The cells will proliferate in the medium until they occupy all Batch-to-batch variability, potential for contamination, and of the available space in the medium (i.e., reach confluence). poor characterization are inevitable. At this stage, the cells have to be subcultured (i.e., passaged) By convention, the passaging or subcultivation of a primary by transferring them to a new vessel with fresh growth cell culture began what is termed cell line or subclone medium to provide more room for continued growth. (Secondary culture). The culture is termed “primary” up to the first subculture. However, not all primary cultures yield cell lines. Instead, the cells of some subcultivated cultures die off slowly. 7 8 2  10/19/2023 Secondary cultures: Finite Cell Line:  Cell lines Following passage of primary cell cultures, some cultures Cell lines are permanently established cell cultures of will survive serial passage before becoming senescent and animal or plant origin (immortal cells) that will proliferate increasingly slow growing. repeatedly and most times indefinitely given appropriate During passage cells with the highest growth capacity fresh medium and space. predominate, resulting in a degree of genotypic and They are population of cells descended from a single cell phenotypic uniformity in the population. and containing the same genetic makeup. Cell lines in this category obtained from normal healthy tissue will continue to display a normal phenotype.  The types are: Finite cell lines Finite cell lines originally derived from embryonic tissues will generally have the potential for a greater number of Continuous cell lines population doublings before the onset of senescence than those derived from adult tissues. 9 10 Good examples of finite cell lines include: Continuous Cell Lines: - Wistar Institute-38 cells (WI38): a diploid human cell These include transformed and immortalized cell lines, line composed of caucasian fibroblast-like fetal lung and are derived directly from tumour material or by cell. Derived from lung tissue of a 3-month-gestation exposure of normal cells to transforming agents. female fetus. Transformation of cells may be due to: - Medical Research Council cell strain 5 (MRC-5): a Exposure to chemical carcinogens diploid cell culture line composed of human fetal lung fibroblast cells, originally derived from embrayonic Ionizing radiation lung tissue of a 14-week-old aborted caucasian male Infection with retroviruses or DNA tumour viruses (or fetus in 1966. viral components) Spontaneous - Both have been used in vaccine production and other research. 11 12 3  10/19/2023 Following transformation, continuous cell lines express Adherent and suspension cultures an altered phenotype. Adherent cultures: Immortalization is one step in the transformation Are said to be anchorage-dependent and attachment to process, and may confer an infinite life span but without a substratum is a prerequisite for proliferation. the alteration in growth control. They are generally subjected to contact inhibition, which Examples of continuous cell lines include means they grow as an adherent monolayer and stop - Vero cells: isolated from kidney epithelial of African dividing when they reach such a density that they touch Green Monkey (Cercopithecus aethiops) in 1962. each other. - HeLa cells: derived from cervical cancer cells of Most cells, with the exception of mature hemopoietic Henrietta Lacks, a 31 year-old African-American cells and transformed cells, grow in this way. woman who died of cervical cancer. 13 14 Cultures in which cells grow and attached to each other Clearly, freely suspended cultures do not require or to a substratum have to be treated by a proteolytic trypsinization. Are, therefore, easier to harvest. enzyme to break the bond between cells and the substratum. The most commonly used enzyme is trypsin (other enzymes, e.g., collagenase, papain, dispase, and pronase, are also used). Suspension cultures: Cells from blood, spleen or bone marrow adhere poorly and thus best cultured as suspension cultures. Suspension cultures are easier to propagate, since subculture only requires dilution with medium. 15 16 4  10/19/2023 Commonly used cell lines of each cell Commonly used cell lines of each cell morphology type morphology type Adherent Cell Lines Suspension Cell Lines Name Species and Tissue of Origin Morphology Name Species and Tissue of Origin Morphology MRC-5 Human Lung Fibroblast HeLa Human Cervix Epithelial NS0 Mouse Myeloma Lymphoblast Vero African Green Monkey Kidney Epithelial U937 Human Hystiocytic Lymphoma Lymphoblast NIH3T3 Mouse Embryo Fibroblast HL60 Human Leukemia Lymphoblast L929 Mouse Connective Tissue Fibroblast Lymphoblast WEHI231 Mouse B-cell Lymphoma CHO Chinese Hamster Ovary Fibroblast YAC1 Mouse Lymphoma Lymphoblast BHK-21 Syrian Hamster Kidney Fibroblast HEK-293 Human Kidney Epithelial U266B1 Human Myeloma Lymphoblast HepG2 Human Liver Epithelial Jurkat Human T-cell Leukemia Lymphoblast BAE-1 Bovine Aorta Endothelial Lymphoblast THP-1 Human Monocyte Leukemia SH-SY5Y Human Neuroblastoma Neuronal 17 18 Morphology of Cells in Culture  Cells in culture can be divided into three basic categories based on their shape and morphology. Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate. Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches. Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface. 19 20 5  10/19/2023 SOURCES OF CELL CULTURES Vertebrate mammals: Predominantly humans or rodents, tissue systems such as: Integument and muscular system e.g. melanocytes, keratinocytes, muscle and fat cells Gastro-intestinal system e.g. epithelium, pancreas, liver, Fibroblast-like cells colon, smooth muscle cells Respiratory system e.g. alveolar and bronchial epithelium Urinary system e.g. kidney tissue Female reproductive system e.g. aminiocyte, chorionic Epithelial-like cells villi, cervical cells etc Male reproductive system e.g. sertoli cells Endocrine system e.g. thyroid, pancreas cells Lymphoblast-like cells 21 22 Osteo-articular system e.g. chondrocytes, synovial Vertebrate non-mammals: these include: cells, osteoblasts Fish, amphibians and birds. Neuronal system e.g. dorsal root ganglion, neuronal, Fish cell lines have been derived from a range of species, glial cells predominantly from embryo tissue, originally for the Cardiovascular system e.g. cardiac myocytes, umbilical growth and study of fish viruses. cord vein endothelial cells Fish cells have a reduced requirement for glutamine and Haemopoietic e,g. progenitor cells, macrophages, T may be incubated at temperatures lower than 37oC. and B cells Other mammals from which cell cultures have been Invertebrate: the cell culture is required for baculovirus derived include rabbit, guinea pig, horse, cattle, dog, growth. cat, monkey etc. Techniques for the maintainance of cultures are similar to those used for mammalian cell culture. Insect cells grow at a lower temperature (27oC) than 23 mammalian cells, and require specialized media. 24 6  10/19/2023 CELL CULTURE MEDIA SUPPLIERS OF CELL CULTURES These provide nutrients in a readily accessible form at It is strongly recommended that cell cultures are optimal pH and osmolality for cell survival and obtained from recognized collections centers such as: proliferation. European Collection of Cell Cultures (ECACC) the temperature, oxygen and carbon dioxide content of the culture are controlled. American Type Culture Collection (ATCC) Media could be: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) - Natural (obtained from natural sources such as cagula or plasma clot, biological fluids, tissue extracts) or These organisations provide pure, authentic, quality- - Artificial (serum-free and protein-free media). controlled cell cultures with information concerning appropriate media and cultivation conditions. In general, all culture media consist of a basal medium, serum and/or other growth factors. 25 26 A] Basal media The components of basal media are: There are several basal media available depending on Balanced salt solution the cell type. Buffering systems For mammalian cell culture, the most widely used are: Carbohydrates or glutamine - Eagle’s medium and derivatives Amino acids - RPM1 medium and derivatives Vitamins, hormones and growth factors Proteins and peptides For insect cell culture commonly used are: Fatty acids and lipids - Mitsuhashi and Maramorosch’s medium Accessory factors such as trace elements and - Schneider’s medium nucleotides. - Grace’s medium 27 28 7  10/19/2023 B] Serum Other supplements include horse serum and chick Fetal bovine serum (FBS) is the most commonly used embryo extract. supplement to basal media. Serum may vary in performance from batch-to-batch, It is a rich source of growth factors, which are effective and should be tested prior to purchase. across a broad range of cell types derived from various species and tissue types. FBS is a major source of viral contaminants in cell The major components of serum include: culture, particularly bovine viral diarrhea virus. - Hormones, - growth factors, - spreading factors, Such viruses may be present in low numbers, and - Binding proteins (albumin, transferrin, vitronectin), require sensitive detection methods such as PCR - Anti-proteases (alpha-1 antitrypsin, alpha-2 technique. macrogiobulin), - vitamins, - minerals, Other common biological contaminants of cell cultures - attachment factors (fibronectin, laminin, fetuin), are bacteria, yeast, molds, mycoplasma etc. - lipids etc. 29 30 Demerits of serum in culture media  Growth factors  The use of serum in culture media is associated with several are supplements to basal media that will promote growth problems including: in the absence of serum or in reduced serum formulations. a. Batch to batch variation, which causes inconsistency in growth promoting properties. Many polypeptides have been characterized as mitogenic b. The high protein content, which can hinder product purification. in vitro for specific mammalian cell lines. c. The risk of contaminants—viruses, mycoplasma, prions. This family includes: d. The risk of transmission of these contaminants to an end product  fibroblast growth factor (FGF: aFGF and bFGF), used by humans, e.g., bovine spongiform encephalopathy (BSE) or  insulin-like growth factor (IGF), mad cow disease.  epithelial growth factor (EGF), e. Interfering with the effect of hormones or growth factors when studying their interaction with cells.  nerve growth factor (NGF), f. Limited availability of fetal calf serum.  platelet-derived growth factor (PDGF), and g. High cost of fetal calf serum, which can account for up to 85% of  transforming growth factor (TGF: alpha and beta). the overall cost of the medium when calculated for large-scale These growth factors are active in the 1–10 ng/mL range. cultures. 31 32 8  10/19/2023 C] Serum-free media D] Chemically Defined Serum-Free Media The development of serum-free media has been driven There is a clear distinction between serum-free media and by the shortcomings of serum, and the desire to simplify chemically defined serum-free media. downstream purification of cell products. Chemically defined media require that all of the components In general, there has been success in the development must be identified and have their exact concentrations of serum-free media for some particular cell types. known. It also allows researchers who are studying in the field of cell Such commercially available media consist of basal physiology (especially extracellular) and/or molecule–cell media supplemented with components such as insulin, interactions to eliminate any variables that may arise due to transferrin, growth factors, hormones, etc. the effects of unknown components in the medium. However, there are no universally applicable serum-free The supplements may be classified into several groups such media. as peptide growth factors, hormones, carrier proteins, hydrolysates, and attachment factors. 33 34  The advantages of using media supplemented with defined growth E] Protein-free media factors and hormones include the following: The media contain non-protein constituents. a. In many cases cells show enhanced growth characteristics in these defined media compared to serum-containing media. Some cells, Compared to serum-supplemented media, use of protein- notably differentiated types, cannot be maintained at all in serum- free media promotes superior cell growth and protein supplemented media and their maintenance in vitro is dependent expression and facilitates downstream purification of any on selected hormones and growth factors. expressed product. b. It is possible to select specific cell types from a mixed population of cells as may be obtained from a primary source. Such cell-specific F] Antibiotics selection requires careful manipulation of the medium The use should be saved for emergency decontamination composition. of irreplaceable cultures, or for the preparation of primary c. Studies of the interaction of hormones or drugs on cultured cells are material from non-sterile sites. made possible by such media. The uncertain composition of serum and the binding effects of the associated proteins would limit such Routine antibiotic use in culture media is not advised due studies in serum supplemented media. to the potential for suppression of low-level bacterial contamination, and selection of antibiotic-resistant strains. 35 36 9  10/19/2023 When antibiotics are used, the following cocktail may be The overall process of water purification for cell culture recommended: preparation involves three or four-stages. - Penicillin (100 IU/mL) to inhibit the growth of Gram- These are: positive bacteria - reverse osmosis (or distillation): provides a relatively - Streptomycin (50mg/mL) to inhibit the growth of Gram- pure water supply to begin the process. negative bacteria - charcoal filtration: remove the majority of organic and - Amphotericin-B (25 mg/mL) as an antifungal agent. inorganic impurities G] Water for Media Preparation - deionization: remove any remainder of trace metals or Due to the delicacy of mammalian cells the presence of trace ions. minerals and/or contaminants will drastically affect the - micropore filtration: remove any possible microbial performance of the culture medium, especially in serum-free contamination in the final water. cultures. In most systems the purified water will be continuously Due to this, specialized water purification systems are used recycled throughout the filtering system, resulting in for water in media preparation. increasingly pure water. 37 38 For the purposes of making culture media the water Phases of cell growth in cell culture should be taken directly from the purification system A typical growth curve for cultured cells displays a sigmoid during media preparation. pattern of proliferation. The growth phases associated with normal cells are defined as: Alternatively, the water can be stored if it is sterilized  Lag Phase – No cells division but adaptation to culture beforehand. conditions.  Logarithmic (Log) Growth Phase – cells actively proliferate This can be accomplished by autoclaving or by (exponential increase in cell density). Cells are also generally micropore filtration (20 mm pore size). passaged at late log phase. Passaging cells too late can lead to overcrowding, apoptosis and senescence. For autoclaving the water should be placed in a suitable  Plateau (or Stationary) Phase – cellular proliferation slows container, such as a pyrex (borosilicate) bottle. down due to cell population becoming confluent.  Decline Phase – cell death predominates in this phase. Cell death is not due to the reduction in nutrients, but to the natural progression of the cellular cycle 39 40 10  10/19/2023 41 42 TISSUE AND ORGAN CULTURING Tissue culture is used as a generic term to include the in vitro cultivation of tissues and organs. Originally, the term is not limited to animal tissues, but includes the in vitro cultivation of plant tissues. TISSUE AND ORGAN CULTURING Tissue culture, just like the cell culture, is the cultivation of small pieces of a multicellular organism (i.e. tissues) in a suitable artificial medium under sterile and appropriate conditions. Officially, the term tissue culture is used when cells are maintained in vitro for more than 24 hours. 43 44 11  10/19/2023 Organ culture refers to a three-dimensional culture of tissue Proper handling of the organ (part or whole) is essential retaining some or all of the histological features of the tissue so that the tissue of the organ would not be damaged in vivo. and disrupted. It is the explantation and cultivation of a part or whole of an The media used for a growing organ culture are animal organ in a sterile controlled medium in the laboratory generally the same as those used for tissue culture. (in vitro) in such a way as to maintain some normal spatial relationships between cells and some normal function. The techniques for organ culture can be classified into: Organ culturing is different from cell culture (where single - those employing a solid medium and cells are cultivated) and tissue culture (where tissue is - those employing liquid medium. involved), even though it was developed from tissue culture methods. Currently, the methods used in organ culture are: What is essential in organ culturing is that the architecture of Hanging drop method, Plasma clot method, Agar gel the tissue of the organ is preserved and is directed towards method, Raft and Grid Method, Intermittent exposure to its development. medium and gas phase. 45 46  There are disadvantages to organ cultures: METHODS USE IN ORGAN CULTURE Organs cannot be propagated so each piece of tissue can Hanging drop method: only be used once, which makes it difficult to assess the The explant of organ is placed on a cover slip and reproducibility of a response. And, of course, the particular covered with clotted lymph. cells of interest may be very small in number in a given piece of tissue so the response produced may be difficult to detect This is inverted over the cavity of a clean grease free and quantify. sterilised glass slide in such a way that the explant is hanging in the cavity. It may not be possible to supply adequate oxygen and nutrients throughout the tissue because of the absence of a The open margins of the cover slip is sealed on to the functioning vascular system, so necrosis of some cells occurs slide and the set up incubated at 37oC and examined fairly rapidly. This problem may be ameliorated to some daily for any growth. extent by keeping the organ in stirred cultures or in roller bottles which alternately provide air and soluble nutrients. 47 48 12  10/19/2023 Plasma clot or Watch glass method: organ rudiments or organs are grown on the surface of a clot consisting of 15 drops of plasma and 5 drops of embryo extract in a watch glass placed on a cotton wool pad. The cotton wool pad is placed in a Petri dish, and moisten time to time to prevent excessive evaporation. A small piece of organ tissue is placed on the top of the plasma clot in the watch glass. The Petri dish may or may not be covered with lid, sealed with paraffin wax and incubated at 37.5oC. Fresh clots is to be provided every 2 to 4 days. 49 50 Agar gel method: Embryonic organs generally grow well on agar, but adult The medium consist of 1% agar in basal salt solution, organ culture will not survive on this medium. chick embryo extracts and horse serum, in the ratio The culture of adult organs or parts from adult animals 7:3:3. is more difficult due to their greater requirement The agar gel medium is kept on embryological watch of oxygen. glass and explant is then transferred on the surface of A variety of adult organs (e.g. the liver) have been agar and sealed with paraffin wax. cultured using special media with special apparatus. Sub-cultured into fresh agar gel every 5 to 7 days. Although the agar does not liquefy, it cannot be added Explant can be examined with stereoscopic microscope. or analysed without transplanting the cultures. The technique is used to study many developmental This disadvantage was overcome by the use of fluid aspects of normal organs and tumours. media combined with a support which prevented the cultures being immersed. 51 52 13  10/19/2023 Raft method: Explants are placed on raft of lens paper or rayon acetate and floated on serum in a watch glass. Floatability of the lens paper or rayon acetate can be enhanced by treatment with silicone. Four or more explants are placed on a single raft. Raft and clot techniques can be combined. The explants in this case are first placed on a suitable raft, which is then kept on a plasma clot. This modification makes media changes easy, and prevents the sinking of explants into liquefied plasma 53 54 Grid method: The chamber is supplied with a mixture of O2 and CO2 to Utilizes 25 mm x 25 mm pieces of a suitable wire mesh meet the high O2 requirements of adult mammalian or perforated stainless steel sheet whose edges are bent organs. to form 4 legs of about 4 mm height. Its widely used to study the growth and differentiation Skeletal tissues are generally placed directly on the grid of adult and embryonic tissues. but softer tissues like glands or skin are first placed on rafts, which are then kept on the grids. The grids themselves are placed in a culture chamber filled with fluid medium up to the grid; 55 56 14  10/19/2023  Intermittent Exposure to Medium and Gas Phase: Importance of cultured organ More recently, a method which provides intermittent Cultured organs can be an alternative for organs from exposure to medium and gas phase was successfully other (living or deceased) people. developed. used for the long-term (4-5months) culture of human This is useful as the availability of transplantable organs adult tissues, including bronchial and mammary epithelium. (derived from other people) is declining in developed countries. In this technique, the explants are attached to the bottom of a plastic culture dish and covered with medium. The dishes are enclosed in an atmosphere-controlled Another advantage is that cultured organs, created using chamber which is filled with an appropriate gas mixture. the patient’s own stem cell allows for organ transplants The chamber is placed on a rocker platform and rocked at and would allow the patient to no longer several cycles per minute during cultivation. require immunosuppressive drugs. 57 58 APPLICATIONS OF CELL CULTURE Production of vaccines Cancer research, which required the study of uncontrolled cell division in cultures. Production of biopharmaceuticals Study the function of the nerve cells Many commercial proteins have been produced by FACTORS AFFECTING CELL animal cell culture and their medical application is being CULTURING evaluated. Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using recombinant DNA technology. The t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders. 59 60 15  10/19/2023 FACTORS AFFECTING CELL CULTURING 1] Physico-Chemical factors Cell culture conditions vary for each cell type. pH of the cell culture medium The consequences of deviating from the culture Most normal mammalian cell lines grow well at pH 7.4, conditions required for a particular cell type can range and there is very little variability among different cell from the expression of aberrant phenotypes to a strains. complete failure of the cell culture. However, some transformed cell lines have been shown to grow better at slightly more acidic environments (pH By controlling the osmotic pressure, media composition, 7.0–7.4), and and environmental parameters, high yields of cells or cell products may be consistently obtained. Some normal fibroblast cell lines prefer slightly more basic environments (pH 7.4–7.7). Certain factors can affect the success of cell culture. Insect cell lines such as Sf9 and Sf21 grow optimally at These factors include: pH 6.2. 61 62  Carbon dioxide tension  Oxygen tension Because the pH of the medium is dependent on the Molecular oxygen is one of the most important variables in delicate balance of dissolved carbon dioxide (CO2) and modern cell culture systems. bicarbonate (HCO3–), changes in the atmospheric CO2 can Fluctuations in its concentration can affect cell growth, alter the pH of the medium. differentiation, signaling, and free radical production. It is necessary to use exogenous CO2 when using media  Osmotic pressure buffered with a CO2-bicarbonate based buffer, especially if the cells are cultured in open dishes or transformed cell Osmotic pressure is of vital importance in biology as the cell’s lines are cultured at high concentrations. membrane is selective toward many of the solutes found in living organisms. While most researchers usually use 5–7% CO2 in air, 4–10% CO2 is common for most cell culture experiments. In hypertonic solution (high solute concentration), cell loses water and shrink to lose its turgidity. However, each medium has a recommended CO2 tension In hypotonic solution (low solute concentration), cell take in and bicarbonate concentration to achieve the correct pH water, swell and eventually rupture. and osmolality. 63 64 16  10/19/2023  Temperature of the cell culture environment Above 30°C, the viability of insect cells decreases, and the The optimal temperature for cell culture largely depends on cells do not recover even after they are returned to 27°C. the body temperature of the host from which the cells were Avian cell lines require 38.5°C for maximum growth. Although isolated, and to a lesser degree on the anatomical variation these cells can also be maintained at 37°C, they will grow in temperature (e.g., temperature of the skin may be lower more slowly. than the temperature of skeletal muscle). Cell lines derived from cold-blooded animals (e.g., Overheating is a more serious problem than under-heating amphibians, cold-water fish) tolerate a wide temperature for cell cultures; therefore, often the temperature in the range between 15°C and 26°C. incubator is set slightly lower than the optimal temperature. Most human and mammalian cell lines are maintained at 36°C to 37°C for optimal growth. Insect cells are cultured at 27°C for optimal growth; they grow more slowly at lower temperatures and at temperatures between 27°C and 30°C. 65 66 2] Physiological factors  Growth factors and hormones  Cell culture Media Cell growth, in general, is increased by various hormones The culture medium is the most important component of the and growth factors that are required nutritionally and culture environment, because it provides the necessary environmentally for cell survival, proliferation, and protein nutrients, growth factors, and hormones for cell growth, as synthesis. well as regulating the pH and the osmotic pressure of the culture. Most of them are present in serum which is added to the culture medium. However, in serum-free media, the growth The addition of serum into the basal medium help to factors and hormone are added directly. provide the various factors (growth factors, and hormones) necessary for cell growth. Growth factors and hormones are groups of polypeptides which control the proliferation and differentiation of normal There is no one suitable medium for all cell type, different cell cells types have unique medium for optimum cell growth and this must be determined before setting up a cell culture. 67 68 17  10/19/2023 BIOPHARMACEUTICALS Biopharmaceuticals (or biologics) are complex pharmaceutical drug products manufactured in, extracted from, or semisynthesised from biological sources and are used for therapeutic or diagnostic purposes. Biotechnological techniques or methods used in the Sources of Biopharmaceuticals manufactured of biopharmaceuticals are: o Recombinant DNA technique o Protein engineering technique o Hybridoma technologies They have biological sources, usually involving live organisms or their active components. 69 70 Biopharmaceuticals include: Sources of Biopharmaceuticals  Recombinant vaccines, whole blood, blood components, Some biopharmaceuticals were previously extracted from allergenics living systems (microbes, animals or humans particularly)  Somatic cells, gene therapies but are now produced by recombinant DNA technologies.  Recombinant therapeutic proteins  Living medicines (i.e. living cells) used in cell therapy Transgenic organisms, especially plants, animals, or microorganisms that have been genetically modified, are  Living tissues potentially used to produce biopharmaceuticals.  Sugars, proteins, nucleic acids, or complex combinations of these substances For example, the therapeutic insulin previously extracted Biopharmaceuticals (or their precursors or components) are from porcine (pigs) pancreatic islets is now produced by isolated from living sources—human, animal, plant, fungal, recombinant DNA technologies in the yeast, Saccharomyces or other microbes. cerevisiae or in a particular strain of E. coli. 71 72 18  10/19/2023 Examples of biopharmaceuticals directly extracted from  Examples of biopharmaceuticals produced by recombinant DNA technology are: living systems are: Blood factors Whole blood and blood components, Tissue plasminogen activators organs and tissue transplants, Hormones stem cells, Hematopoietic growth factors (granulocyte/macrophage antibodies for passive immunization, colony-stimulating factor, monocyte/macrophage colony- fecal microbiota stimulating factor, stem cell factor, erythropoietin etc. human reproductive cells Interferons, interleukin-based products Vaccines Thrombolytic agent Monoclonal antibodies Tumor necrosis factors 73 Therapeutic enzymes etc 74 Advantages of biopharmaceuticals Highly effective and specific Fewer side effects They are not carcinogenic They are safe Easy commercial production Production of biopharmaceuticals, quality control and validation. 75 76 19  10/19/2023 Production of biopharmaceuticals - Product (Active ingredient – API) is created either by cell Production of biopharmaceutical products involves many culture, recombinant techniques or fermentation, first in different, complex and lengthy steps. small laboratory bottles/plates then introduced into These include: small bio-reactor and then into larger bio-reactors which  Manufacturing can be several stories tall.  Purification  Purification:  Formulation - After production, the product must be purify (i.e.  Final dosage form preparation removal of cells from the cellular nutrients they grew in and the byproducts) to become the bulk product.  Manufacturing: - The bulk product can be sold as it is or processed further - Ordering of raw materials to its final formulation - testing the raw materials to be sure they meet quality  Formulation: standards - Several operations are required to get bulk product into - Sterilization of equipment and production materials to its final form. Involves mixing with other substances such avoid microbial contamination of cell cultures 77 as fillers needed in the final form 78  Final dosage form preparation: The vast majority of biopharmaceutical products currently on - The formulated preparation is made into its final form, the market are produced by recombinant DNA technology in dispensed into containers, labeled and packaged. either E. coli or Chinese Hamster ovary (CHO) cell lines. Most monoclonal antibody based products are produced by hybridoma technology, although the technical methodology now exists to facilitate production of antigen-binding antibody fragments by recombinant means. E. coli represents a popular recombinant expression system for a number of reasons: - E. coli is easy to culture and grows rapidly. - Its genetic characteristics are exceedingly well- characterized and reliable standard protocols for its genetic manipulation have been developed. 79 80 20  10/19/2023 - Appropriate fermentation technology is well established, Recombinant proteins may be expressed in a number of and high expression levels of recombinant proteins are other microbial systems which do contain the enzymatic generally attained. activities to facilitate post-translational processing. E. coli, however, does display some disadvantages as a recombinant production system. Various proteins have been expressed, both in yeast - Recombinant proteins generally accumulate (particularly Saccharomyces cerevisiae) and fungi intracellularly, complicating downstream processing. (especially various species of Aspergilli). - Often more critically, E. coli lacks the ability to glycosylate proteins (or carry out any other post-translational While such microorganisms are capable of glycosylating modifications). recombinant therapeutic proteins, the pattern of glycosylation usually differs to that associated with such - Many proteins of therapeutic interest are naturally glycosylated and lack of the carbohydrate component can, proteins when expressed naturally in the human body. potentially, adversely affect its biological activity, solubility, or in vivo half-life. 81 82 Such microbial expression systems exhibit a number of More recently, a number of recombinant therapeutic characteristic advantages and disadvantages in terms of proteins produced in various animal cell lines have gained recombinant protein production. marketing approval. Thus far, few recombinant biopharmaceuticals developed are Chinese hamster ovary (CHO) cells and baby hamster produced in either yeast or fungal systems. kidney (BHK) cell lines have become popular recombinant production systems. Two approved biopharmaceuticals produced in Saccharomyces cerevisiae are: Patterns of glycosylation associated with recombinant glycoprotein biopharmaceuticals produced in CHO and BHK  Recombinant hirudin (Refludan) an anticoagulant resembles most closely the native glycosylation pattern marketed by Behringwerke AG). when the protein is produced naturally in the human body.  Recombinant hepatitis B surface antigen, incorporated The production of recombinant therapeutic proteins (tissue into various combination vaccines by SmithKline plasminogen activator, arantitrypsin, interleukin 2, factor IX Beecham. etc.) in the milk of transgenic animals has also gained much 83 publicity over the last few years. 84 21  10/19/2023 Biopharmaceuticals may be produced in bioreactors of various configurations (including photo-bioreactors) from: Microbial propagation system have the disadvantage of intracellular product expression,  Microbial cells (e.g., recombinant E. coli or yeast cultures),  Mammalian cell lines and plant cell cultures and cell needs to be lysed to obtain product and cell debris is  Moss plants. released which required purification of the product. Important issues of concern are: A cell culture propagation system, on the other hand, has the  cost of production (low-volume, high-purity products are advantage of extracellular expression which significantly desirable) and simplifies the process of purification requirements.  microbial contamination (by bacteria, viruses, mycoplasma). In addition, cell culture is capable of producing the biologically active pharmaceutical ingredient (API) in a form Alternative platforms of production which are being tested that is more usable by the human body, which also simplifies include whole plants (plant-made pharmaceuticals). downstream processing and improves product potency. 85 86 Production of biopharmaceuticals can be by Batch or As bio-processing is a wet operation requiring frequent Continuous culture method. cleaning, bioprocess equipment is typically designed and constructed in a manner that facilitates external cleaning Batch processing has historically dominated biotechnology and sanitization. manufacturing because of available technologies, risk aversion, and perceived regulatory difficulties associated with Biotechnology manufacturing operations are typically continuous culture. categorized as upstream or downstream processes. However, emerging technology has opened up many options Upstream bio-processing refers to all of the manufacturing to make continuous processing more feasible in drug processes required to produce the biologically active manufacturing. pharmaceutical ingredients (APIs), including:  inoculum preparation, Bioprocess development for future facilities is expected to make greater use of continuous processing to enable more  bioconversion (via fermentation or cell culture), and efficient manufacturing on a small scale.  harvest steps (via centrifugation or filtration). 87 88 22  10/19/2023 Downstream bio-processing refers to all of the processing Typical Biotechnology manufacturing required for the API to meet purity and quality operations requirements, including product recovery, purification, and polishing steps (via chromatography and filtration). After downstream processing, the purified drug substance is formulated (sometimes also conjugated) and filled into a bulk container for storage in a stable form. 89 90 Equipment cleaning and bioburden control The CIP rinsing operations are typically the largest user of Traditional biomanufacturing in stainless steel equipment compendial water in a facility, so system drainability and requires facility infrastructure for cleaning and sterilization. minimizing holdup are important for economical and sustainable water use. Centralized clean-in-place (CIP) systems are designed to This places some constraints on facility design, whereby it is prepare and distribute cleaning solutions throughout the plant often desirable to locate CIP systems in mechanical space to facilitate cleaning of equipment in situ. near the equipment to be cleaned. In multilevel facilities, it may be advantageous to locate CIP Single-pass systems may be required for cleaning areas with areas in the space above or below the process to facilitate potential biohazard exposure. the drainability of distribution piping. The design of distribution loops for CIP is critical because it For some small bioprocess systems, cleaning may be requires piping that is correctly sloped and free of dead legs accomplished with temporary connections to portable CIP (i.e., pockets with stagnant areas). systems or through disassembly and cleaning-out-of-place (COP). 91 92 23  10/19/2023 QUALITY CONTROL AND VALIDATION OF Biopharmaceutical companies must conform to the stringent BIOPHARMACEUTICALS Good Manufacturing Practice (GMP) regulations established The term “Quality Control”, refers to the sum total of all by the FDA/NAFDAC. procedures undertaken during production to ensure the correct quality of the final product. The three department that ensure quality of products are: The quality of a product may deviate from the standard o Quality Control (QC): Samples and test raw materials and required during and after production, so carrying out the product during many stages of the manufacturing analysis is important in order to determine if the quality of process. the final product falls within the required standard. o Quality Assurance (QA): Ensures product quality by setting Analysis done to determine quality of products may include: up and checking the systems of standard operating Chemical, Physical and sometimes Microbiological analysis procedures (SOPs) if followed and documentation done or evaluation. during manufacturing process. Deviation from SOP must be documented and approved by QA department. Product quality must be ensured throughout the product life - All processes and checks done must be written down, if cycle, from raw materials down to the final product. not written down then it never happened. 93 94 o Validation: this proves that a manufacturing process will Biotechnology is an applied science that is generally consistently produce the product to predefined regarded as new and rapidly evolving advanced technology. specifications. - The operation of every part of the plant that affects product Biotechnology facilities are fundamentally different from other pharmaceutical manufacturing facilities because they quality must be validated. are required to harness the inherent complexities and - If a manufacturing process is changed or if a new product is variability of living things. introduced, all processes and equipment that affect quality must be validated. Biologics are large and complex molecules that are difficult, - Validation scientists and engineers have extensive if not impossible, to characterize completely. experience because they must be familiar with the In contrast to traditional pharmaceutical facilities that regulations. manufacture small-molecule drugs via chemical synthesis, biotechnology facilities produce large-molecule biologics (i.e., proteins with a molecular weight greater than 5,000 Da). 95 96 24  10/19/2023 Most biological drugs have the ability to generate a Bioburden control is required from the initial manufacturing significant immune response in the human body. steps through the final fill. Relatively small changes to the manufacturing process can Aseptic processing (i.e., process operations that are devoid have a significant effect on the efficacy or immunogenicity of measurable bioburden) is required for many of these of the drug. steps. In most cases, biologics are also unstable over time and Most biopharmaceutical products are delivered as a require special handling and storage to protect the product parenteral (i.e., injectable) dosage form, whereby the drug is from degradation. injected directly into the bloodstream; In comparison with small-molecule drugs, some manufacturing processes are more susceptible to microbial Thus, the level of contamination prevention and bioburden contamination. control required is much more stringent than that required for oral or topical dosage forms. 97 98 Bioburden is a particular issue with injectable drugs for the Product contamination can come from airborne following three key reasons: particulates, raw materials, utility systems, product contact  Endotoxins created by Gram-negative microbial materials, and other products manufactured in the same contaminates can elicit an immune response with facility. unwanted side effects (e.g., inflammation, fever, internal Therefore, the prevention of product contamination is the bleeding, and septic shock). predominant principle driving every aspect in the design process of biotech facilities.  Exotoxins from environmental bacteria, which are not routinely monitored, can cause cell culture death and are highly toxic to humans in small quantities.  Bacterial action on the product may cause unwanted variants by clipping protein chains or changing the glycosylation pattern (neither of which may be readily detectable by analytical testing). 99 100 25  10/19/2023 Biosafety-classification and levels Biosafety levels (BSL) are used to identify the protective measures needed in a laboratory setting to protect workers, the environment, and the public. There are many ways to combine equipment, practices, and laboratory design features to achieve appropriate Biosafety-classification and levels biosafety and biocontainment. These are determined through biological risk assessments specifically conducted for each experimental protocol. 101 102  Risk assessments are conducted by evaluating: At any given biosafety level, there will be strict requirements for laboratory design, personal protective equipment, and The way in which the infectious agents or toxin is biosafety equipment to be used. transmitted Standard Microbiological Practices are required at all The ability of the infectious agents or toxin to cause biosafety levels. disease Activities and projects conducted in biological laboratories The activities performed in the laboratory, the safety are categorized by biosafety level. equipment and design elements present in the laboratory The four biosafety levels (BSLs) are BSL-1, BSL-2, BSL-3, and The availability of preventive medical countermeasures or BSL-4. treatment BSL-4 is the highest (maximum) level of containment. The health and training of the laboratory worker. 103 104 26  10/19/2023 Biosafety Level 1 (BSL-1) Biosafety Level 2 (BSL-2) BSL-1 labs are used to study infectious agents or toxins BSL-2 laboratories are used to study moderate-risk infectious not known to consistently cause disease in healthy agents or toxins that pose a risk if accidentally inhaled, adults. E.g. Saccharomyces cerevisiae, E. coli K-12, non- swallowed, or exposed to the skin. E.g. Hepatitis A virus, infectious bacteria. Streptococcus pyogenes, Salmonella species, Borrella burgdorferi (Lyme disease). They follow basic safety procedures, called Standard Design requirements for BSL-2 laboratories include hand Microbiological Practices and require no special washing sinks, eye washing stations in case of accidents, and equipment or design features. doors that close automatically and lock. BSL-2 labs must also have access to equipment that can Standard engineering controls in BSL-1 laboratories decontaminate laboratory waste, including an incinerator, an include easily cleaned surfaces that are able to autoclave, and/or another method, depending on the withstand the basic chemicals used in the laboratory. biological risk assessment. 105 106 Biosafety Level 3 (BSL-3) Other engineered safety features include the use of two BSL-3 laboratories are used to study infectious agents or self-closing or interlocked doors, sealed windows and toxins that may be transmitted through the air and cause wall surfaces, and filtered ventilation systems. potentially lethal infection through inhalation exposure. E.g. Yersinia pestis, Mycobacterium tuberculosis, SARS, rabies BSL-3 labs must also have access to equipment that can virus, West Nile Virus, Hantaviruses. decontaminate laboratory waste, including an incinerator, an autoclave, and/or another method, Researchers perform all experiments in biosafety depending on the biological risk assessment. cabinets that use carefully controlled air flow or sealed enclosures to prevent infection. BSL-3 laboratories are designed to be easily decontaminated and uses controlled, or “directional,” air flow to ensure that air flows from non-laboratory areas (such as the hallway) into laboratory areas as an additional safety measure. 107 108 27  10/19/2023 Biosafety Level 4 (BSL-4) Cabinet laboratory – all work with infectious agents or toxins BSL-4 laboratories are used to study infectious agents or is done in a Class III Biosafety Cabinet with very carefully toxins that pose a high risk of aerosol-transmitted laboratory designed procedures to contain any potential infections and life-threatening disease for which no vaccine contamination. or therapy is available. E.g. Ebola virus, smallpox virus The laboratories incorporate all BSL 3 features and occupy In addition, the laboratory space is designed to also prevent safe, isolated zones within a larger building or may be contamination of other spaces. housed in a separate, dedicated building. Access to BSL-4 laboratories is carefully controlled and requires significant training. There are two types of BSL-4 laboratories: - Cabinet laboratory - Suit laboratory 109 110 Biological safety cabinet (BSC) Class I safety cabinet, which is the most basic one, Biological safety cabinet (BSC) is an engineering control provides personnel and environmental protection only. intended to protect laboratory workers, laboratory When properly used Biological safety cabinets have environment and work materials from exposure to infectious been shown to be highly effective in reducing or biohazardous aerosols and splashes. laboratory-acquired infections and cross-contaminations Such aerosols and splashes may be generated while of cultures. manipulating materials containing infectious agents, such as primary cultures, stocks and diagnostic specimens. Safety cabinets have to be certified periodically for their efficiency. There are three kinds of safety cabinets: Classes I, II, and III. Various field tests are performed to verify air flows, HEPA filter integrity, containment of contaminated Class II and Class III biological safety cabinets provide: cabinet air etc. personnel, environmental as well as product protection. 111 112 28  10/19/2023 Class I Biological safety cabinet Class I Biological Safety Cabinet (BSC) is the first designed and simple BSC which provides personnel and environmental protection but not product protection. Unsterilized room air is drawn over the work surface through the front opening at a minimum velocity of 0.38 m/s. The front opening also allows the operator’s arms to reach the work surface inside the cabinet while he or she observes the work surface through a glass window. The window can also be fully raised to provide access to the work surface for cleaning or other purposes. 113 114 The directional flow of air whisks aerosol particles that may be generated on the work surface away from the laboratory worker and is then discharged from the BSC through a HEPA filter. Class I BSCs are suitable for work with Risk Group 1 (RG1), Risk Group 2 (RG2), and Risk Group 3 (RG3) biological material. HEPA filter traps 99.9% of particles of ≥ 0.3 µm in diameter. This enables the HEPA filter to effectively trap all known infectious agents and ensure that only microbe-free exhaust air is discharged from the cabinet and/or recirculated in the work surface. 115 116 29  10/19/2023 Class II Biological Safety Cabinet (BSC) About 90% of all biosafety cabinets installed are Type A2 cabinets. A Class II Biological Safety Cabinet (BSC) is a ventilated cabinet, which provides personnel, product and There is a limited need for Class II Type B biological safety environmental protection. cabinets. In addition, Class II Type B biological safety cabinets require It is commonly found in clinical and research laboratories very specific installation and operating conditions to function working with infectious agents in Risk Groups 2, 3 and 4 (if correctly. positive-pressure suits are used) or with tissue culture. The working mechanism of Class II BSCs differs according to There are four types (A1, A2, B1, and B2 ) of Class II BSCs. its types. It has an open front with inward airflow for personnel The main differences between the types are the ratio of air protection, downward HEPA filtered laminar airflow over the exhausted from the BSC to the air that is recirculated within work surface for product protection and HEPA filtered the BSC, and the type of exhaust system present. exhausted air for environmental protection. 117 118 The room air and recirculated air are HEPA filtered before flowing downwards over the work area. Class II BSCs can be exhausted into the containment zone or directly to the outside atmosphere through a thimble or hard- ducted connection depending on the types. The amount of air that recirculates or exhausts depends on the types. 119 120 30  10/19/2023 Class III Biological Safety Cabinet (BSC) The Class III BSCs should have an attached pass-through Class III BSC provides the highest level of personnel box that is sterilizable and is equipped with HEPA- protection and is used for Risk Group 4 agents. filtered exhaust. It is suitable for work in Biosafety Level 3 and 4 laboratories. The Class III cabinet may be connected to a double-door This type of cabinet is totally enclosed and is tested under autoclave used to decontaminate all materials entering pressure to ensure that no particles can leak from it into the or exiting the cabinet. room. Supply air is HEPA-filtered and exhaust air is discharged to atmosphere through two HEPA filters. The operator access the work surface by means of heavy- duty rubber gloves which form part of the cabinet. 121 122 Biohazards and containment Biohazards, also known as biological hazards are biological substances that pose a threat to the health of living organisms, primarily that of humans. Biohazards refers to organisms or organic matter produced by these organisms that are harmful to human health. This include viruses, bacteria, fungi, parasites and protein. Biohazard symbol (Designed in 1966 by Charles Baldwin, an environmental-health engineer) Serves to warn : 123 124 31  10/19/2023 Levels of Biohazard The United States Centers for Disease Control and Prevention (CDC) categorizes various diseases in levels of biohazard, Level 1 being minimum risk and Level 4 being extreme risk.  Biohazard Level 1: Bacteria and viruses including Bacillus subtilis, Canine hepatitis, Escherichia coli, varicella (chicken pox), as well as some cell cultures and non-infectious bacteria. At this level precautions against the biohazardous materials in question are minimal, most likely involving gloves and some sort of facial protection. 125 126  Biohazard Level 2:  Biohazard Level 4: Bacteria and viruses that cause only mild diseases to humans Viruses and bacteria that cause severe to fatal disease in or are difficult to contract via aerosol in a lab setting, such as humans, and for which vaccines or other treatments are not hepatitis A, B and C, some influenza A strains, salmonella,