A  Review: Production of Monoclonal Antibody

 

Kachariya Brijesh*,Vihar Gadhvi,  Amit Gupta, Komal Roopchandani, Nirav Patel

Department of Pharmaceutics, Mahatma Gandhi College of Pharmaceutical Sciences, ISI-15 (A) RIICO Institutional Area, Sitapura, Tonk Road, Jaipur-302022 (Rajasthan).

*Corresponding Author E-mail:- brijeshkachariya@gmail.com

 

 

ABSTRACT:

Monoclonal antibodies (mAbs) are currently used for many diagnostic and therapeutic applications. The high demand for these biopharmaceuticals has led to the development of large-scale manufacturing processes, with productivity improvements being mainly achieved by optimization of bioreactor systems. However, more recently, the early steps of production, previous to bioreactor culture, have been presented as alternative areas where productivity enhancements can be achieved. Selection of the most suitable clones is also a critical step that can be improved, by including variables other than the expression level, which is still the common practice. Furthermore, strategies of cell engineering, although still mostly based on trial-and-error experimentation and not in standard protocols, hold great interest to improve cell growth and productivity, as well as product quality in the future.  Monoclonal antibodies can be designed that have customized affinityand specificity against drugs of abuse,in vivo pharmacokineticcharacteristics can be tailored to suit specific clinicalapplications (eg, long-acting for relapse prevention, or short-acting for overdose). Passive immunization with antibodies against drugs of abuse has several advantages over active immunization, but because large doses of monoclonal antibodies may be needed for each patient, efficient antibody production technology is essential.

 

KEYWORDS: Introduction, advantages, disadvantages, production of monoclonal antibody.

 

 


INTRODUCTION:

Antibody treatments are in preclinical and clinical development for addressing a wide range of medical problems caused by drug abuse. These treatments include vaccination to help prevent relapse to nicotine addiction, 1,2 and monoclonal antibodies (mAb) to (1) treat overdose from phencyclidine or methamphetamine, 3-5 or (2) prevent relapse to methamphetamine abuse. 6 A considerable obstacle for mAb therapy (eg, immunoglobulin G [IgG]), antigen binding fragment [Fab]; single chain antigen binding fragment [scFv]; is that discovering potentially important mAb medications and then producing sufficient quantities for rigorous preclinical or clinical testing takes a while. This is a major hurdle that must be overcome if this new technology is to be taken to clinics in a timely manner.

 

Because the early-stage manufacturing and formulation of mAb-based medications is both technologically challenging and expensive, this review will examine the production of these therapies at the levels needed for preclinical in vivo studies (eg, 0.5-10 g), with a special emphasis on alternative production systems, including plants.

 

 


Fig. 1.Integration of relevant information necessary for evaluation of antibody PK and PD properties and clinical dose selection.POM proofof-mechanism, POP proof-of-principle, POC proof-of-concept biomarkers

 

 


ADVANTAGES OF MAB THERAPY:

Passive immunotherapy with mAb has important advantages over active immunization.

1)     Significantly larger doses of mAb can be administered, and protection is immediate.

2)     The duration of action of mAb is more predictable than antibody generated by active immunization and likely to be related to the biological half-life of the mAb. For example, currently approved mAb medications for treating other disease processes (eg, cancer) have halflives of up to 28 days in humans.7

3) unlike with active immunizations, there is no immunological memory of the abused drug and the possibility of unexpected cross reactivity with endogenous ligands is less likely with mAb.

4)     Another advantage of mAb medications is the ability to preselect for the affinity and specificity of the antibody, and thereby have the medication parameters be constant from one production lot to the next.

5)     The use of MoAbs in cancer treatment is focused on the idea of selectively targeting tumor cells that express tumor-associated antigen8, with the aim to specifically antagonize receptor signaling pathways, which are essential for proliferation, survival, and migration of tumor cells.

6)     Speed and specificity and sensitivity of assay.

7)     Antigen or immunogen need not be pure.

8)     Selection helps to identify the right clones against the specification.

 

DISADVANTAGES OF MAB THERAPY:

1)     Antibody-based therapies could potentially save lives and reduce the crippling effects of chronic drug abuse, but they will not be a magic bullet to cure addiction. In many ways the problems associated with treating drug abuse are analogous to the problems associated with treating a chronic infectious disease (eg, HIV).

2)     For instance, certain individuals and segments of the population are more susceptible to infectious diseases, and the best way to prevent epidemics is to stop the spread of the disease from individual to individual.

3)     By analogy, we need medical prevention to aid individuals and groups who are at an increased risk for addiction, and we need specific medications for treating key individuals who are important vectors for spreading drug abuse in the population.

4)     The 3 major problems are the high cost, the risk of toxicity, and the potential for allergic-type reactions. The current cost for mAb medications for treating cancer and other health problems is thousands of dollars per month.

5)     Immune complex formation accelerates clearance, reduces serum levels, and impairs targeting of the therapeutic antibodies. 7,9

6)     System is only well developed for mouse and rat and not for other animals.

 

Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies10-23

Production of monoclonal antibodies involves in vivo or in vitro procedures or combinations thereof. Before production of antibodies by either method, hybrid cells that will produce the antibodies are generated. The steps in producing those cells are outlined below (figure 1). The generation of mAb-producing cells requires the use of animals, usually mice. The procedure yields a cell line capable of producing one type of antibody protein for a long period. A tumor from this “immortal” cell line is called a hybridoma.

 

No method of generating a hybridoma that avoids the use of animals has been found. It has also been possible to genetically replace much of the mouse mAb-producing genes with human sequences, reducing the immunogenicity of mAb destinedfor clinical use in humans.Development of the hybridoma technology has reduced the number of animals (mice, rabbits, and so on) required to produce a given antibody but with a decrease in animal welfare when the ascites method is used.

 

Step 1: Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells:

Mice are immunized with an antigen that is prepared for injection either by emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. Intact cells, whole membranes, and microorganisms are sometimes used as immunogens. In almost all laboratories, mice are used to produce the desired antibodies. In general, mice are immunized every 2-3 weeks but the immunization protocols vary amonginvestigators. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.

 

Step 2: Screening of Mice for Antibody Production:

After several weeks of immunization, blood samples are obtained from mice for measurement of serum antibodies. Several human techniques have been developed for collection of small volumes of blood from mice. Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry. If the antibody titer is high, cell fusion can be performed. If the titer is too low, mice can be boosted until an adequate response is achieved, as determined by repeated blood sampling. When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.

In part, prepared from Current Protocols in Molecular Biology. Ed: Frederick M. Ausubel, 1998.

 

Step 3: Preparation of Myeloma Cells:

Fusing antibody-producing spleen cells, which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth. Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion.1 A week before cell fusion, myeloma cells are grown in 8-azaguanine. Cells must have high viability and rapid growth. The HAT medium allows only the fused cells to survive in culture.

 

Step 4: Fusion of Myeloma Cells with Immune Spleen Cells:

Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse. As noted in step 3, only fused cells will grow in the special selection medium. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells.

 



Shaded are as represent mouse use.

 

Step 5: Cloning of Hybridoma Cell Lines by “Limiting Dilution” or Expansion and Stabilization of Clones by Ascites Production:

At this step new, small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time. Cloning by “limiting dilution” at this time ensures that a majority of wells each contain at most a single clone. Considerable judgment is necessary at this stage to select hybridomas capable of expansion versus total loss of the cell fusion product due to under population or inadequate in vitro growth at high dilution. In some instances, the secreted antibodies are toxic to fragile cells maintained in vitro. Optimizing the mouse ascites expansion method at this stage can save the cells. Also, it is the experience of many that a brief period of growth by the mouse ascites method produces cell lines that at later in vitro and in vivo stages show enhanced hardiness and optimal antibody production. 

 

Invitro production of monoclonal antibody:

A major advantage of using mAb rather than polyclonal antiserum is the potential availability of almost infinite quantities of a specific monoclonal antibody directed toward a single epitope (the part of an antigen molecule that is responsible for specific antigen-antibody interaction). In general, mAb are found either in the medium supporting the growth of a hybridoma in vitro or in ascitic fluid from a mouse inoculated with the hybridoma. mAb can be purified from either of the two sources but are often used as is in media or in ascitic fluid. In vitro methods should be used for final production of mAb when this is reasonable and practical. These devices vary in the facilities required for their operation, the amount of operator training required, the complexity of operating procedures, final concentration of antibody achieved, cost, and fluid volume accommodated. The cost of A major advantage additional equipment should be considered in the cost of invitro production methods.

 

Batch Tissue-Culture Methods:

The simplest approach for producing mAb in vitro is to grow the hybridoma cultures in batches and purify the mAb from the culture medium. Fetal bovine serum is used in most tissue-culture media and contains bovine immunoglobulin at about 50 µg/ml. The use of such serum in hybridoma culture medium can account for a substantial fraction of the immunoglobulins present in the culture fluids.To avoid contamination with bovine immunoglobulin, several companies have developed serum-free media specifically formulated to support the growth of hybridoma cell lines. In most cases, hybridomas growing in 10% fetal calf serum (FCS) can be adapted within four passages (8-12 days) to grow in less than 1% FCS or in FCS-free media. However, this adaptation can take much longer and in 3-5% of the cases the hybridoma will never adapt to the low FCS media. After this adaptation, cell cultures are allowed to incubate in commonly used tissue-culture flasks under standard growth conditions for about 10 dys;mab is then harvested from the medium.

The above approach yields mAb at concentrations that are typically below 20 µg/ml. Methods that increase the concentration of dissolved oxygen in the medium may increase cell viability and the density at which the cells grow and thus increase mAb concentration Some of those methods use spinner flasks and roller bottles that keep the culture medium in constant circulation and thus permit nutrients and gases to distribute more evenly in large volumes of cell-culture medium.The gas-permeable bag (available through Baxter and Diagnostic Chemicals), a fairly recent development, increases concentrations of dissolved gas by allowing gases to pass through the wall of the culture container. All these methods can increase productivity substantially, but antibody concentrations remain in the range of a few micrograms per milliliter.

 

The above approach yields mAb at concentrations that are typically below 20 mg/ml. Methods that increase the concentration of dissolved oxygen in the medium may increase cell viability and the density at which the cells grow and thus increase mAb concentration.

 

CONCLUSION:

mAb and mAb engineered fragments present potentially effective therapies to treat drug abuse. The high affinity and exquisite specifi city of these proteins to their drug targets and their customizable pharmacokinetic properties make them very attractive therapeutic candidates. However, like many protein-based pharmaceuticals, these medications are difficult to move from the bench to the clinic because of the large amounts needed for therapeutic doses. New advances in molecular technology and our understanding of these complex systems are beginning to overcome these issues and will eventually help stimulate cost-effective protein-based therapies for the treatment of drug abuse, so that these medications can become widely available.

 

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Received on 27.02.2013          Modified on 21.03.2013

Accepted on 13.04.2013         © RJPT All right reserved

Research J. Pharm. and Tech 6(7): July 2013; Page 701-705