Propose of this report is to discuss the downstream process for monoclonal antibody. Downstream process for monoclonal antibody includes several processes, such as Centrifugation, Filtration, Precipitation, Chromatography, Viral Clearance, Drying & Crystallization etc and which will refer to the flowsheet of the entire downstream processing later on. The key subject for this report is to discuss the filtration process in more detail as one of the downstream process for monoclonal antibody.
1.1 Monoclonal Antibodies
Antibodies are basically proteins and it is produced by the B lymphocytes. In immune system, B lymphocytes response to foreign proteins which is then called antigens. The function of antibodies is combined to the antigen, once the antigen molecules recognized the pathogen, the antigen will destroyed pathogen by phagocytes. In a specific part of the antigen combined with the antibody, which part of the area or cell called epitope while the epitope has a short amino acid sequence, which the antibodies are able to recognize. (Karnik, 1998)
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Two characteristic of the relationship between antibody and epitope are the key issues of the use of monoclonal antibodies as a molecular implement. Two characteristics are specificity and sufficiency, they could be describe as the antibody only combines to its specific epitope and the epitope could combines to the antibody by itself respectively. (Karnik, 1998)
Due to the antibodies are proteins and it consisted four polypeptide chains in their structure. They form a quaternary structure which is similar to a Y shape which is shown in Figure . (Karnik, 1998)
Figure . An immunoglobulin G antibody molecule (IgG). (Thomas, 2007)
In an organism synthesizes, each B cell has only one type of antibody. In organism includes different types of B cell in an entire population. At the same time, the organism also consist the B cell respective to their antibodies. Each antibody produced in response to the different antigens since the organism has been exposed. From this point, the better solution is to produce a large amount of a single antibody. A method is needed to cultivate a population of B cell turn into a single ancestral B cell. Therefore, this method of population of B cell could be produced a single kind of antibody and this population of cells could be then describe as monoclonal. This population of B cells will then produced the antibodies and they are called monoclonal antibodies (mAb). (Karnik, 1998)
Monoclonal antibodies have extraordinary specificity and purity and they are constituent of the immune system. The function of monoclonal antibodies is they can be able to recognize and combine to a specific antigen. (Hart, 1996)
1.2 Application of Monoclonal Antibodies
Monoclonal antibodies have a wide range of applications in medical, academic and commercial. Monoclonal antibodies can be used in several diagnostic tests, which can be able to detect a small amount of hormones, drugs and toxins. For example, pregnancy test kits and diagnosis of Acquired Immune Deficiency Syndrome (AIDS) by the Enzyme-Linked Immunosorbent Assay test (ELISA). In the other hand, monoclonal antibodies are also used in the radioimmunodetection and radioimmunotherapy of cancer, a new technique can aim the cancerous cells' membranes. Strains of a single pathogen can be easily to classify by using monoclonal antibodies, for example, Neisseria gonorrhoeae. However, some researchers can be able to follow the specific molecules or cells in an organism by using monoclonal antibodies. (Karnik, 1998)
1.3 Advantages and disadvantage of Monoclonal Antibodies
Five major advantages of Monoclonal Antibodies are as follows:
Monoclonal antibodies can be produced in a unlimited quantities of monospecific reagent. (Sheehan, 2010)
Monoclonal antibodies are antibodies of a single antigen molecule which has the same affinity for the functions of the same effect. (Sheehan, 2010)
Single hybridoma product reacts or response the same epitope from antigens. (Sheehan, 2010)
Monoclonal antibodies do not need to be characteristics or pure and ultimately do not need to produced a large amount of antibodies.( immunizing antigen) (Sheehan, 2010)
The monoclonal antibodies can select a specific epitope to have its own specificities and be able to generate the antibodies which are against more extensive range of antigenic determinants (Sheehan, 2010)
Five disadvantages of Monoclonal antibodies are affinity, effector functions, specificity, cross-reactions and time and effort commitment, each parameter will described as following respectively: (Sheehan, 2010)
Always on Time
Marked to Standard
Monoclonal antibodies have an average affinity and normally, they are lower than polyclonal antibodies. Polyclonal antibodies are the antibodies which have obtained from different B cell. (Sheehan, 2010)
Since the antibody is monoclonal, the antibody may not produce or generate the response of desired biologic. (Sheehan, 2010)
Monoclonal antibodies may lose reactivity with antigen when it is against conformational epitopes on native proteins. (Sheehan, 2010)
Monoclonal antibodies occasionally display unexpected cross-reactions with irrelevant antigens. (Sheehan, 2010)
Monoclonal antibodies procedure requires a very long time and large effort commitment. (Sheehan, 2010)
According to the flowsheet of the monoclonal antibody downstream process, filtration process has been used twice, where the filtration process followed by the centrifugation process/ affinity process and the other filtration is needed once more to follow when the virus clearance process has used
The definition of filtration is to separate the substance in solid state to from liquid states. Filtration is usually used when the amount of solid is much less than liquid, which in terms of a mixture could be filtrate when the mixture is majority liquid state.
Filtration could be used in the membrane process and it has classified into three methods by using their different driving forces and pore sizes, which are micro-filtration, ultra-filtration and reverse osmosis. The relationship between different forces and their pore sizes is the bigger the pore size the smaller driving force is required. In the other words, the pore size of micro-filtration is the largest (0.02-10 microns), which has compared to the other methods while reverse osmosis method has non-porous membrane. Due to the bigger size of the pore size, the more driving force is needed to apply to the micro-filtration which is about 0-10 bar pressure, i.e. for ultra-filtration, the pore size is 0.001-0.2 microns and the driving force is 0-100 bar pressure.
2.2 Ultra-filtration and Reverse Osmosis
In this report is to discuss the filtration as one of the downstream process in monoclonal antibody. Following this, two methods are normally used in the downstream process and they are ultra-filtration and reverse osmosis. Normally, ultra-filtration and reverse osmosis filtrate or separate the soluble proteins, salt ions or organic acid.
Due to the different pore sizes and driving forces applied in these two method, this resulting the different product which can be produced. For example, the products for the ultra-filtration are albumin, immunoglobulins, enzymes, other proteins and large oligopeptides. However, for the reverse osmosis method, the products are such as gluose, small peptides and salts.
Ultra-filtration and reverse osmosis is the process of membrane separation; this separation required more than two fluids. The separation effects when several fluids pass through the membrane and at the same time, some of the fluid retained up to the membrane. In the other words, in different method of filtration has different pore sizes and the pore size of the membrane has function of selective certain molecules can pass through. Normally, the molecule that has retained at the membrane is termed the solutes. The target of membrane separation is to concentration the solutes as much as possible. Membrane separation can be proceeding in low energy and in mild conditions. Three main issues should be considered in membrane separation which is the different size of solute (the solvent should be at least a factor of 10), the material should be has a function of heat sensitive and required the concentration of a dilute feed.
In this report will concentrate to discuss the ultra-filtration in detailed since it is more likely relevant to the monoclonal antibodies downstream process.
2.3 Ultra-filtration (UF)
As a cell separation, the ultra-filtration is a popular technique of antibiotic manufacturers while it has been used in the clarification of bacitracin broths. (Stowell, Bailey, & Winstanley, 1950) Ultra-filtration is a separation process to separate the macromolecules by using certain amount of pressure. (Bouchard, Carreau, Matsuurab, & Sourirajanb, 1994)
Ultra-filtration is the way to purify the solutes in the downstream process. The product is buffer to replace into the formulation buffer. Use the ultra-filtration or dia-filtration setup to achieve monoclonal antibodies purification. The meaning of the purification is to follows the removal of insolubles. The trans-membrane pressure applied, the type of membrane used, the cross-flow rate and concentration that dia-filtration is made could be template for all monoclonal antibodies. With several formulations demanding the use of very high concentrations of protein and issues of viscosity and product collection, this resultant the certain molecules can occur. (Shukla, Hubbard, Tresse, Guhan, & Low, 2007)
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Three particular characteristics in ultra-filtration process, which are the process is using high cross flow, the process is controlled by the membrane and depends on the membrane geometry in the factual equipment. In the following information will give a brief idea of each characteristic. (Belter & Cussler, 1988)
2.3.1 Cross Flow
Normally, the ultra-filtration involves a large flow across through the surface of the membrane or perpendicular to the flux across the membrane, i.e. the flow is fundamental for the ultra-filtration. During the process, when the solid particles are ultra-filtrated, the cross flow will reduces the evolution of a filter cake which would inhibit the ultra filtration process. In the other hand, when the macromolecular solution is ultrs-filtrated, the cross flow minimizes accumulation of macromolecules which near the membrane's surface. In this situation the accumulation is then called '' concentration polarization'', increase the osmotic pressure, therefore, the flow through the membrane will be reduces. (Belter & Cussler, 1988)
Belter & Cussler, 1998 states that ''Ultra-filtration is critically dependent on the membrane. In this sense, it is the antithesis of conventional filtration, where the choice of filter medium usually has little effect on flow through the cake''. The reason behind of the importance of the membrane for the ultra-filtration is because the cross flow minimize the concentration polarization or the cake formation.
In an ultra-filtration module, solutions majority flows tangentially to the surface of the membrane, since there is the pressure difference across the membrane, some part of the solution flow through the membrane. Some solute is incompletely or completely retained behind the membrane, the solute accumulates at the surface of the membrane which developing a concentration boundary layer alongside the ultra-filtration channel. This is called ''concentration polarization''.
Figure . Ultra-filtration membranes. (Belter & Cussler, 1988)
From Figure is introduced the three common types of membranes. The first type of the membrane (referred to Figure A.) is made by spreading a thin layer of organic solution on glass, water or an inert support. This type of membrane has a 80% porosity and their average pore sizes range is between about 0.1 and 1.0 Âµm. (Belter & Cussler, 1988)
The second type of membrane (referred to Figure B.) is made from polypropylene. The porosity of this membrane is around 35% which is less than the first type of membrane. The second type of membrane must be primed with water-alcohol mixtures before ultra-filtration of aqueous solution since they are hydrophobic. However, they are the best obtainable membrane for heart-lung machines, but for ultra-filtration they are not a very common type of membrane that has been used. (Belter & Cussler, 1988)
Figure C is the third type of the membrane, which has a lowest porosity about 3%. The arrangement of the pore is the most monodisperse. This membrane have similar permeability to the other type of membrane since the membrane are 10 times thinner. (Belter & Cussler, 1988)
Figure . Membrane geometries for ultra-filtration. (Belter & Cussler, 1988)
Two key issues to make ultra-filtration effective which are high cross flow and materials for the membrane. For the Figure a, Since this membrane consisted the smallest area of the volume which has compared to the other type of membrane, this resultant to gives a low ultra-filtration fluxes per volume but is the easiest to repair or clean due to the shape. Following this, this geometric is easier to disassemble for replacement of imperfect membrane or for cleaning. (Belter & Cussler, 1988)
Figure b, shell and tube has lower area per volume therefore lower ultra-filtration fluxes than the spiral wound (Figure c) and hollow fiber (Figure d) geometries. At this case, it is harder to clean the device than the flat sheet type device (Figure a). (Belter & Cussler, 1988)
Figure c, spiral wound consist a larger are per volume than flat sheet geometrics (Figure a) and tubular geometrics (Figure b) therefore higher ultra-filtration fluxes produced. Due to its shape is much harder to clean or repair, when the membrane fails, the solution is often discarded. (Belter & Cussler, 1988)
Figure d, hollow fiber and tubular (Figure b) geometries are similar to each other but the scale is much different while the diameter of the hollow fiber is much smaller than the tubular's the ultra-filtration modules of hollow fiber and flat sheet devices are antithesis due to the hollow fiber could be able to give a huge area per volume, hence it produces the highest ultra-filtration fluxes per volume. The disadvantage of fiber device is too hard to clean, furthermore, once a few fibers failed, the entire module must usually be disposed. (Belter & Cussler, 1988)
All the type of devices required additional equipment which is shown in Figure , which means each device must consist a holding tank, a membrane module and a feed pump. The function of the pump is to increased the speed of the feed to past through the membrane, in the record, the pump could produced a speed at least 10 times higher than the flux pass through the membrane by itself. Some material will retained behind and will be recycled. (Belter & Cussler, 1988)
Figure . Batch ultra-filtration. (Belter & Cussler, 1988)
Four different types of geometrics equipments of ultra-filtration has different advantage and disadvantage, beyond this point, spiral wound (Figure c,) and hollow fiber( Figure d) will be good advice to prefiltration before starting the ultra-filtration process, since these two geometrics could be produced higher ultra-filtration fluxes. In the other hand, other particles such as protein might irreversibly absorb to the surface of membrane, these resultants the fouling in the membrane devices. Membrane usually blocked in the devices and this resulting by the concentration polarization. Therefore to clean the membrane device is a very important issue to consider in certain situation when choose the specific membrane equipment for ultra-filtration. (Belter & Cussler, 1988)))
2.4 Major Use for Ultra-filtration
Three major uses for the ultra-filtration which are Solute Concentration, Solute Fractionation or Clarification and Solute Desalting or Purification, these will be described as respectively as follows.
Solute concentration can be increased by using ultra-filtration membrane technique. The aim of the filtrate is to scavenging the macromolecules. Macromolecules are larger than the pore of the retentive membrane following this, the micro-solute then can be eliminated convectively with the solvent. (VIVASCIENCE)
When separating the samples into different size of the components. This also provided that the macromolecular size difference up to 10X molecular weight difference. The permeating solute stays as its initial concentration during filtration while the retained macromolecules will be concentrated. (VIVASCIENCE)
A solution could be purified by three parameters which are non-aqueous solvents, salts and the material which has a low molecular weight. Multiple solvent exchangers will purify the macromolecules from the solutes which has contaminated. The most effectual solution is to add the solvent to the solution to remove the microsolutes while its being ultrfiltered, which has the same rate of the filtration. This is then called Dia-filtration. (VIVASCIENCE)
2.5 Ultra-filtration Applies in Monoclonal Antibodies
In Koticha et al's, 2006 research, a newly developed processing method of monoclonal antibodies in hybridoma supernatants is introduced. A simply method could be reduced the processing time to obtain the pure monoclonal antibodies. Two steps are involved in this process which is clarification and concentration. Firstly micro-filtration is used to clarification of the hybridoma supernatant. Secondly, the monoclonal antibodies needs to concentration by using ultra-filtration. (Koticha, Barbagallo, & Rapiejko, 2006) In the other words, the contaminants could be eliminate from supernatant which permits macromolecules(less than 100,000 molecular weight) to pass through the membrane and retained the monoclonal antibodies and which is concentrated and purified more than 25- fold. (Saha, Case, & Wong, 1992) The monoclonal antibodies are then more purified on protein A and protein G beads. Following this, ultra-filtration is used again at the end to desalt and buffer-exchange the monoclonal antibodies. (Koticha, Barbagallo, & Rapiejko, 2006)The application of ultra-filtration is cell removal from the cephamycin C broths within large scale. Since, ultra-filtration is normally used in large scale; therefore it reduced its capital cost which will be the advantage of this process. Two issues that affect the capital cost of the ultra-filtration, which are low energy required and low cost for the maintenance. (Stowell, Bailey, & Winstanley, 1950)
The ultra-filtration is then suitable to apply into the downstream process in monoclonal antibody while one of the products from ultra-filtration is immunoglobulins and this product is related to the monoclonal antibodies.
Immunoglobulins are also known as antibodies in the blood and the immunoglobulins test is usually taken to measure the level of immunoglobulins. Antibodies are produced by plasma cell and it is design specify to destroy or inhibit foreign pathogen. Antibodies are acquired immune response which is designing specific to a pathogen and effectively.
The antibodies could be classified into five types which are, IgA, IgG, IgM, IgE and IgD
IgA found in external secretion such as saliva, tears, intestinal and bronchial mucus and breast milk, it disables pathogen before they reach the internal environment. (Silverthorn, 2010) IgA antibodies present 10-15% of antibodies. Nevertheless, few people do not have or produce IgA antibodies in their blood (Web MD, 2008)
IgG is found in75% of plasma antibody and it produces in secondary immune response. Maternal IgGs cross the placental membrane and provide infant immunity, its' function is activate complement. (Silverthorn, 2010) This type of antibody can recognize the harmful particles. The size of the IgG antibodies are the smallest which has compared to the other antibodies, however, IgG antibodies are most common antibody which is presented in the human blood and in mathematical record, IgG are presenting about 75-80% of all antibodies in the body. (Web MD, 2008)
IgM is found in plasma; it is involves in primary immune response and react to blood group antigens. It is a activate complement (Silverthorn, 2010) the size of the IgM is the largest size antibodies. In the body, 5-10% of IgM antibodies present of all antibodies. (Web MD, 2008)
IgE locate in plasma, it associated with allergic responses, when mast cell receptors bind with IgEs and antigen and the mast cells degranulate and release chemical mediators such as histamine. (Silverthorn, 2010) IgE might occur in allergic reactions to some medicines and milk etc.The level of IgE antibodies are normally high, when the people with allergies. (Web MD, 2008)
IgD is found on the surface of B-lymphocytes, but how they work is unclear (Silverthorn, 2010)
Figure . Process flow of mAb purification from hybridoma cells. (Koticha, Barbagallo, & Rapiejko, 2006)
In Koticha et al's, 2006 research, two different types of method has been demonstrated and compared to each other; they are traditional method and UF-based method (referred to section 2.4). These methods have the same function to filtrate the virus or the solid particles out of the monoclonal antibodies before the process goes into the chromatography process. In traditional method, two key stages involved, which are centrifugation and ammonium sulfate precipitation centrifugation dialysis. 22-26 hours required to operate this process. In the other hand, in UF- based method, micro-filtration and ultra-filtration has been required. The total time to operate this process is about 40 minutes, which is much faster than the traditional method. In this project, the best and the most efficiency method is need to be chosen. When compare the traditional method and the UF- based method, the UF- based method is the most efficiency, easier to use, resultants that higher recovery and for the economic has comparable price. Therefore, in this project, the UF- based method is choose only to operate before the chromatography process begins.
According to the flowsheet, the filtration process needs to operate twice, where is before the chromatography process and after viral clearance process. Hence, two filtrations need to operate in different stages of the downstream processing of monoclonal antibodies. These two filtrations will be indicated as filtration A and filtration B respectively. The decision has been made for the filtration A by using the US-based method. For the filtration B, only ultra-filtration is taking place to operate after the viral clearance process, since the viral clearance process has evacuated the majority of the virus in the monoclonal antibodies.