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Design and operation of typical aseptic & aerobic fermentation process



 Fermentation technology .


As a further and necessary disclaimer. and nothing herein should be considered “engineering consulting” for your specific project. Things that can be overlooked in non-sterile manufacturing will present significant issues with Aseptic. (Someone once said Engineers really aren’t boring people. The second is that there is clearer direction in regulatory directives as to fundamental scope requirements. However. as well as latest guidance documents. This course provides an introduction to Aseptic operations in the Biopharmaceutical industry. you must evaluate each project on its own merits.FUNDAMENTALS OF ASEPTIC PHARMACEUTICAL PROCESS Introduction Aseptic Pharmaceutical Engineering is perhaps the most interesting to an engineer compared to other pharma/biotech projects. Due to the ever-changing regulatory environment.) There are two primary reasons it is a favorite. refer particularly to FDA’s 21 CFR parts 210 and 211. and clearer expectations as to the end product. . general practices will be discussed without specific reference to the predominant FDA and EU guidances as much as possible. The goal is to provide the student with a well-rounded introduction to Aseptic operations. The first is the technical challenge. There is less to debate. they just like boring things. Engineers like to begin with a firm scope.

When the product can be terminally sterilized (autoclaving the most common method). an Aseptic operation maintains acceptable sterility at critical steps of the manufacturing process (when sterile filtration or other means are not possible) and filling operations (when terminal sterilization is not an option). Aseptic processing is not necessary.) . Aseptic processing is common for parenterals (injectible drugs. Unlike products that are terminally sterilized (the preferred method by major regulatory agencies).What is Aseptic? Aseptic simply means there are no microorganisms that can cause infection in the patient.

Salmanella. coli. A simple example of this is normal drinking water.M. there is no ill effect.” 2 These may include bacteria such as E.) and bypass the defense mechanisms. If you drink safe water. and Neisseria as well as other pathogens. and cause pain. Solutions that have different osmalarity can cause damage to red blood cells or tissue irritation. Especially careful formulation of parenterals is also important.V. parenterals are injected intramuscularly (I. you could get extremely sick. Haemophilus.Whether produced in an Aseptic manner or terminally sterilized. usually refers to fever caused by bacterial endotoxins. . But if you were to inject the same water with a syringe.) or intravenously (I.”)1 An Endotoxin is “cell wall debris (lip polysaccharide) from Gram-negative bacteria. parenterals must be sterile in their final form to avoid problems for the patient. Shigella. Products that are not sterile may contain pyrogens (“an agent capable of inducing an increase in body temperature. A parenteral is formulated to have the same osmolarity of the blood (approximately 300 milliosmoles per liter or mOsmol/L). Whereas drugs such as OSD’s (Oral Solid Dosage) do not require sterility since the body’s natural defense mechanisms engage after ingestion. Pseudomonas.

therefore. .It is critical. As well. The product 2. And mostly. to produce such products in an environment that mitigates contamination and to a rigid spec. people. Packaging components and materials 5. As we will study later. it is important to design Aseptic areas that minimize the number and effort of workers. extreme care is required to protect the product from the natural contamination of the worker. The environment/HVAC 3. Equipment 4. Sources of contamination include the following: 1.

(Be aware of the EU designations since they are different for at-rest and in-operation.) . However.000 cleanrooms that will be rendered sterile later. and D). B. It is common to produce products in class 100. the EU classifies by letters A. or ISO designations.The Manufacturing Process If the product can be sterilized prior to fill/finish1. The class may be referred to as other designations by regulatory agencies (for example. the manufacturing process is not required to be in an Aseptic environment. care must be taken to minimize thebioburden – we wouldn’t want to manufacture the product in the parking lot. A cleanroom class is measured by the quantity of viable (produced from living matter) and non-viable particles. C.

they still can be an “extraneous contaminate”3 to the product. and can contaminate it biologically by acting as a microbial vehicle.) However. which we will discuss later.What does the class mean quantitatively? For class 100. Class 100.Aseptic and less critical activities. for example. in the direct Aseptic area (exposed sterile product) the class must be 100. (There is no specific general cleanroom classification requirement for all non-sterile drugs.000 in a millimeter). there must be less than 100. Although the particulates may be nonviable (non-living).5 micron and larger particles in a cubic foot of air (there are 25. and 1. See Figure 1 below for comparative sizes of particulate.000 particles of 0. .000 can be used for non.000.400 microns in an inch.

Figure 1 Comparative Particle Sizes .

However. also called “Critical Areas. Such filters should be validated that they repeatedly remove viable microorganisms from the sterilized process stream. it is protected in a sterile state and packaged. and certain process steps must be undertaken in closed or class 100 cleanroom environments (this means there are no more than 100 particles 0. These filters should be capable of a 10-3 SAL (we will discuss SAL later). some products cannot be sterilized prior to filling. valves should be steam sterilizable in some applications. Tanks holding or processing sterile products should be maintained in a pressurized state or otherwise sealed to prevent contamination from microbes.5 micron and larger in a cubic foot of air). irradiation. Once the product is sterilized.Typical sterilization techniques of the product prior to fill include heat.4 Filters should be pre/post-bubble tested to confirm integrity.22-micron filter (or less) which is sufficient to remove most bacteria and molds (but may let viruses and mycoplasmas through).” . Filters are tested to remove 107 Brevundimonas diminuta microorganisms per cm2 while producing a sterile effluent. and most commonly filtration through a 0.

THE FILL/FINISH PROCESS OVERVIEW Here we reach the most critical steps of the process as it relates to maintaining sterility in a typical application.) Further. operational aspects are crucial. Such monitoring should coverall shifts. equipment. (Remote particle monitoring for nonviables is a preferred solution in addition to settling plates for viables. However. Means will be required to monitor environmental conditions on an on-going basis. At the point of entry into the Aseptic fill room. the product must be and remain sterile. Design must be accomplished such that it is robust enough to minimize problems that lead to contamination. and closures. . time limits should be established for each processing phase. As well. containers. viable testing can include surfaces. and especially sterile product contact items.5 Obviously. Instead. no amount of monitoring will guarantee sterility. the operation will rely on Validated procedures to keep the product from risk. such as room finishes.

You are probably familiar with these. it should contain a preservative to permit multiple use). That isn’t a fashion statement – it is so a visual examination can confirm the liquid is colorless and sufficiently transparent. One thing you might have noticed when getting that dreaded shot is that the containers are translucent. typically holding 100 ml or more) in bags or bottles (type II glass). extracting the product. made of type I glass for SVP’s (Small Volume Parenterals). and LVP’s (Large Volume Parenterals. which are commonly used when receiving a vaccination (inserting a syringe needle in the top stopper.There are several finished forms of Aseptic produced products. ampoules (a sealed glass container with a long neck that must be broken off). . What are the typical finshed forms? (See Figure 2). The most common are glass vials (single and multi-dose. if multidose.) Other forms include pre-filled syringes.

Ampoule. syringe. vial .

. However. which becomes the finished form. Also included are ointments and creams.The form of the final product can be powder or liquid. Powder can be produced by a sterile crystallization process prior to filling the vials. labeling.2 a Critical Area. The puncture is quickly sealed. this tends to have a less accurate fill than liquids. Once the stopper is installed. The important thing to remember is that during the fill process (while the product is exposed) the immediate environment must be a class 100 Cleanroom. as well as offer other material handling challenges. and cartooning can be in a lower grade environment. the over seal (arguably). Also. Filtering reduces microbiological concentration of the product supply solution rendering it sterile as discussed previously. disposable filling equipment is available. A final liquid form is often created by adding WFI (Water for Injection) to the compound and then filtered. maintaining Aseptic integrity. The vials can be filled with liquid. Another promising technology is filling sterile liquid into vials with needles that are pre-sterilized/pre-sealed. Sterile nitrogen is used to reduce the concentration of oxygen during the filling operation.

particles. eliminating the possibility of terminal sterilization. sublimation. biological materials require freeze-drying to better stabilize them. During the fill process. Here is how it occurs. Often.To add additional stability to products when required. Therefore. liquid can be freeze-dried after being placed in the vials but prior to complete stoppering. it must be maintained in an Aseptic Class 100 environment until lyophilized and finally sealed. Sublimation involves vaporizing a solid and condensing it without its having passed through a liquid state. Lyophilization consists of three distinct processes – freezing. Certain products. A medical provider will reconstitute the product with a suitable solvent (usually WFI) prior to use. such as proteins. or cells into the surrounding medium. This can present a challenge that must be thought through when designing an operation. and blood products. the vial is partially closed. pharmaceuticals. Desorption involves “the release of adsorbed molecules. and desorption. Freeze drying is called Lyophilization. Freeze drying is often used for vaccines. don’t react well to heat.”3 .

) CIP can be problematic in the Aseptic area. (Note: Sterilize is different from Sanitize. whereas Sanitize reduces viable organisms to an acceptable level.4 The key to controlling bioburden is to adequately clean. Sterilize means to destroy viable organisms and spores.cleaned. autoclaving is preferred for product contact parts. however. Filling equipment must be designed to be cleanable. and sanitized/sterilized. Therefore. dry. CIP/SIP is sometimes used (Clean in place/Sterilize in place). . Endotoxins on equipment surfaces can be inactivated by heat. so proceed with caution. and removed by cleaning procedures. it is essential that the design of such equipment facilitate this by being easy to be assembled/disassembled . and store equipment.Careful consideration must be given to all Aseptic equipment. Moist heat is common for sterilization.

Another finish form/technology is BFS (Blow/Fill/Seal). . and sealing it in a single operation. For a comparative overview/PFD (Process Flow Diagram) for typical approaches. see Figure 3. This involves forming a parison (a tubular form) from a plastic polymer resin. at the present this method cannot accommodate Lyophilization. However. inflating it. filling it.



usually an already compromised patient. dry heat ovens. . also know as sterile filling. Pasteurization does not provide sterility. but can reduce the contamination of fungi. Plasma products did have. still remains one of the most critical processes in biopharmaceutical manufacturing. The history of aseptic fill/finish processing is relatively recent with the sterility requirements for injectables being established in the 1920s and large scale biological manufacturing of blood and plasma products during WWII. Ethylene Oxide. a post-fill pasteurization process of low heat treatment of 60°C for 10 hours. and irradiation. either Cobalt 60 Gamma or E Beam.INTRODUCTION Aseptic filling of sterile drugs. Thus the need to utilize an aseptic process to fill certain biologicals. the stability of the aseptic filled drugs will be affected by steam autoclave. pharmaceuticals and biotechnology drugs. Unlike terminal sterilized filled drugs. Anti-fungicidal reagents were also added to parenteral drugs to help mitigate the contamination that was occurring with early aseptic processing. and some products still use. There are only indirect safeguards for the sterility of the filled drug after it is stoppered and capped in the clean room. This is due to its highly technique driven processes and the potential safety impact to the end user.

Micro contamination is very small. and sterilized filling components. and the surfaces that look clean and sterile may in fact not be. the FDA published its Concept Paper: Aseptic Guidelines in 2003 [15]. the fill/finish equipment system. since micro contamination is not readily visible. There is also the perception issue for aseptic fill/finish. sterilized product. equipment and controls. detailed procedures. Thus the aseptic fill/finish processes are highly dependent on technique. the Parenteral Drug Association (PDA) published its Aseptic Validation Technical Report in 1981 [8].Then in an effort to help improve consistency in aseptic processing. which is another reason for the many safeguards that I will discuss shortly. Aseptic filling is an aseptic process that requires the close coordination and complex interaction between personnel. clean room and support facilities. The International Society of Pharmaceutical Engineering (ISPE) published its Sterile Facilities as part of their Guidelines Series in 1999 [14]. Recently. This was followed by the Food & Drug Administration (FDA) in 1987 with its Aseptic Processing Guidelines [1]. .


creates a healthy. Aerobic fermentation actually is a misnomer. beer and acetic acid vinegar (such as apple cider vinegar). . flavorful vinegar with the correct pH. In the presence of Oxygen (aerobic) “cellular respiration. the goal of lactofermentation. need oxygen in the “primary” or first stage of fermentation. The bacteria involved in fermentation actually prefer to use Oxygen. When creating acetic vinegar.” not fermentation. Wine. because it is more energy efficient. fermentation by definition is anaerobic.Anaerobic fermentation is a complicated process which is 100% natural and is carried out on micro-organisms. Read on to know some interesting facts about this process. However. for example. Aerobic fermentation means that oxygen is present. “cellular respiration” does NOTproduce lactic-acid. exposing the surface of the vinegar to as much oxygen as possible. occurs.

Both stirred tank type and air lift type aerobic bioreactor can operate in closed type of batch mode at times in continuous mode. The reactor content is well agitated with impeller and are agitated with the help of stabilized air. The stirrers are used for complete mixing of medium. Adequate supply of air helps in easy achievement of air bubbles agitation. Air lift type: The aerobic fermenters can behave as an air lift type where the mechanical force is absent. . an aerobic condition in closed type or batch mode operation is preferred along with submerged cultures. When there is continuous supply of cells the flow reactor can operate in continuous mode. In modern process.TYPES OF AEROBIC FERMENTERS Stirred tank type: The aerobic fermenters can behave as a stirred tank type where in motor is driven by stirrers with the help of mechanical force. The continuous supply of cells helps in easy release of product in to fermentation medium.