Upstream process is the first step of bioprocess from early cell isolation and cultivation, to cell banking and culture development of the cells until final harvest where the desired quantity is reached. Since this is the early stage of bioprocessing, the quality of the product is of critical importance. Sustainability of this procedure must also be considered. Thus, compliance to cGMP standards is required. One of the measurable requirement is to warrant that from this stage to the filling and packaging must ensure sterility of the process. These parameters will include the use of equipment like bioreactors and cell therapy aseptic isolators, presence of a clean room, and only certified biotechnologist and technicians will be allowed to handle the delicate living organisms.
Materials needed by the biomanufacturing process to make product are weighed and measured in a traceable, controlled, and sanitary area prior to their use. Optimally, raw materials are received into the manufacturing facility in packaging which is suitable for cGMP processing areas (e.g., glass or plastic with no cardboard or other fiber shedding materials). In cases where larger containers of bulk material are received, the exact amount required for the production batch will need to be removed from the bulk, packaged appropriately, and then transferred to the point of use. The dispensing room is the location where raw materials for use in the production process are weighed or measured.
It is critical to mitigate risk or cross-contamination of one material to another during the dispensing process. Per regulatory guidelines, non-animal-derived and animal-derived raw materials must be segregated to reduce risk of exposure to adventitious viruses. To achieve this, dispensing booths are often dedicated to either animal-derived material or non-animal derived material. For an extra level of precaution these material can be weighed in separate rooms.
Vessel used during the biomanufacturing process and all associated piping/hoses must be free of any foreign substances prior to use. Foreign substances include cell debris, medium, cleaning chemicals, and even the target protein form a prior batch. As most bioreactors are multi-use (and may be multi-product), any substances inadvertently left behind can contaminate the next batch. Product left behind from the previous run could encourage microbial growth. Since APIs are drugs intended for introduction into patients, it is critical that the manufactured product be pure. Clean in Place (CIP) and Steam in Place (SIP) are validated cleaning and sterilization procedures that ensure the bioreactor is safe for use.
CIP involves automatic cleaning of processing equipment, vessels, piping, and in-line devices with minimal manual setup and shutdown and little or no operator intervention during cleaning. Sprayballs are used to clean the inner surfaces of the tank during CIP. Sprayballs are located within a vessel and have precisely located holes that ensure the cleaning solutions contact the entire interior surface of the tank.
Chemicals used in CIP are usually strong base solutions (such as potassium hydroxide) that are applied over all surfaces using the sprayballs, followed by rinsing and the application of a strong acid such as phosphoric acid. These substances are all rinsed away by a final WFI spray so that no substances remain. The CIP skid uses conductivity to measure the content of cleaning fluids to ensure proper cleaning. Rinsewater must meet the threshold for conductivity to be considered clean. The acceptance of the CIP is based on reaching required conductivity or resistivity setpoints (targeted values) for specific durations; the setpoints ensure the consistent bioburden and TOC reduction is achieved.
SIP occurs when bioreactor vessels and piping are sterilized with clean steam to establish a sterile boundary. The sterile boundary defines the areas that are kept sterile during cell culture operations and includes the bioreactor vessel and all process piping up to either a major isolation valve or gas filter. Sterilization is critical to prevent contamination. After sterilization is complete, the system must remain pressurized to maintain sterility. If the system pulls a vacuum, the SIP is repeated.
Fermentation is a biochemical process employing selected micro-organisms and microbiological technologies to produce a chemical product. Batch fermentation processes involve three basic steps: inoculum and seed preparation, fermentation, and product recovery or isolation (Theodore and McGuinn 1992). Inoculum preparation begins with a spore sample from a microbial strain. The strain is selectively cultured, purified and grown using a battery of microbiological techniques to produce the desired product. The spores of the microbial strain are activated with water and nutrients in warm conditions. Cells from the culture are grown through a series of agar plates, test tubes and flasks under controlled environmental conditions to create a dense suspension.
The cells are transferred to a seed tank for further growth. The seed tank is a small fermentation vessel designed to optimize the growth of the inoculum. The cells from the seed tank are charged to a steam sterilized production fermentor. Sterilized nutrients and purified water are added to the vessel to begin the fermentation. During aerobic fermentation, the contents of the fermentor are heated, agitated and aerated by a perforated pipe or sparger, maintaining an optimum air flow rate and temperature. After the biochemical reactions are complete, the fermentation broth is filtered to remove the micro-organisms, or mycelia. The drug product, which may be present in the filtrate or within the mycelia, is recovered by various steps, such as solvent extraction, precipitation, ion exchange and absorption.
Solvents used for extracting the product generally can be recovered; however, small portions remain in the process wastewater, depending upon their solubility and the design of the process equipment. Precipitation is a method to separate the drug product from the aqueous broth. The drug product is filtered from the broth and extracted from the solid residues. Copper and zinc are common precipitating agents in this process. Ion exchange or adsorption removes the product from the broth by chemical reaction with solid materials, such as resins or activated carbon. The drug product is recovered from the solid phase by a solvent which may be recovered by evaporation.
Chemical synthesis processes use organic and inorganic chemicals in batch operations to produce drug substances with unique physical and pharmacological properties. Typically, a series of chemical reactions are performed in multi-purpose reactors and the products are isolated by extraction, crystallization and filtration (Kroschwitz 1992). The finished products are usually dried, milled and blended. Organic synthesis plants, process equipment and utilities are comparable in the pharmaceutical and fine chemical industries.
Pharmaceutical chemistry is becoming increasingly complex with multi-step processing, where the product from one step becomes a starting material for the next step, until the finished drug product is synthesized. Bulk chemicals which are intermediates of the finished product may be transferred between organic synthesis plants for various technical, financial and legal considerations. Most intermediates and products are produced in a series of batch reactions on a campaign basis. Manufacturing processes operate for discrete periods of time, before materials, equipment and utilities are changed to prepare for a new process. Many organic synthesis plants in the pharmaceutical industry are designed to maximize their operating flexibility, due to the diversity and complexity of modern medicinal chemistry. This is achieved by constructing facilities and installing process equipment that can be modified for new manufacturing processes, in addition to their utility requirements.
Multi-purpose reactors are the primary processing equipment in chemical synthesis operations. They are reinforced pressure vessels with stainless, glass or metal alloy linings. The nature of chemical reactions and physical properties of materials (e.g., reactive, corrosive, flammable) determine the design, features and construction of reactors. Multi-purpose reactors have external shells and internal coils which are filled with cooling water, steam or chemicals with special heat-transfer properties. The reactor shell is heated or cooled, based upon the requirements of the chemical reactions. Multi-purpose reactors have agitators, baffles and many inlets and outlets connecting them to other process vessels, equipment and bulk chemical supplies. Temperature-, pressure- and weight-sensing instruments are installed to measure and control the chemical process in the reactor. Reactors may be operated at high pressures or low vacuums, depending upon their engineering design and features and the requirements of the process chemistry.
Heat exchangers are connected to reactors to heat or cool the reaction and condense solvent vapours when they are heated above their boiling point, creating a reflux or recycling of the condensed vapours. Air pollution control devices (e.g., scrubbers and impingers) can be connected to the exhaust vents on process vessels, reducing gas, vapour and dust emissions (EPA 1993). Volatile solvents and toxic chemicals may be released to the workplace or atmosphere, unless they are controlled during the reaction by heat exchangers or air control devices. Some solvents and reactants are difficult to condense, absorb or adsorb in air control devices (e.g., methylene chloride and chloroform) due to their chemical and physical properties.
Bulk chemical products are recovered or isolated by separation, purification and filtration operations. Typically, these products are contained in mother liquors, as dissolved or suspended solids in a solvent mixture. The mother liquors may be transferred between process vessels or equipment in temporary or permanent pipes or hoses, by pumps, pressurized inert gases, vacuum or gravity. Transferring materials is a concern due to the rates of reaction, critical temperatures or pressures, features of processing equipment and potential for leaks and spills. Special precautions to minimize static electricity are required when processes use or generate flammable gases and liquids. Charging flammable liquids through submerged dip tubes and grounding and bonding conductive materials and maintaining inert atmospheres inside process equipment reduce the risk of a fire or explosion (Crowl and Louvar 1990).
Organic synthesis reactions may create major process safety risks from highly hazardous materials, fire, explosion or uncontrolled chemical reactions which impact the community surrounding the plant. Process safety can be very complex in organic synthesis. It is addressed in several ways: by examining the dynamics of chemical reactions, properties of highly hazardous materials, design, operation and maintenance of equipment and utilities, training of operating and engineering staff, and emergency preparedness and response of the facility and local community. Technical guidance is available on process hazard analysis and management activities to reduce the risks of chemical synthesis operations (Crowl and Louvar 1990; Kroschwitz 1992).
Large volumes of natural materials, such as plant and animal matter, may be processed to extract substances which are pharmacologically active (Gennaro 1990; Swarbick and Boylan 1996). In each step of the process, the volumes of materials are reduced by a series of batch processes, until the final drug product is obtained. Typically, processes are performed in campaigns lasting a few weeks, until the desired quantity of finished product is obtained. Solvents are used to remove insoluble fats and oils, thereby extracting the finished drug substance. The pH (acidity) of the extraction solution and waste products can be adjusted by neutralizing them with strong acids and bases. Metal compounds frequently serve as precipitating agents, and phenol compounds as disinfectants.