Monoclonal Antibody Production using Animal Cell Culture

monoclonal antibody Antibody Production exploitation Animal electric cell CultureIntroductionMonoclonal antibodies, in biomedical research, argon used as reagents in diagnosis and discourse of ailments like cancer and infections 1. It has been almost century their introduction, mAbs argon still turnd from splenocytes fused to myeloma cells 2. The antibodies are produced by obtaining cell lines from animals immunized with substance to be studied. To produce the cell lines, B cells obtained from immunized mice are fused with myeloma (immortalized) cells 13. For production of desired monoclonal antibody antibodies, the cells should pose in one of the two ways injecting the peritoneal cavity of mouse (known as in vivo regularity or mice ascites rule) or by in vitro method (Tissue culture method). Further mouse ascites fluid or supported of tissue culture is processed and monoclonal antibody of desired concentration and purity is obtained (figure1) 1. Mice ascites method is pr eferred as it is familiar, properly chthonicstood and extensively used in laboratories in comparison to tissue culture method which is time consuming, expensive and laborious and ails to charge required amount of antibodies13. Presently, twenty two monoclonal antibodies for transplantation, oncology, infectious, cardiovascular and chronic inflammatory disease have been approved by FDA 3. Strict guidelines has been fortuneup by IACUC for use of animal for mAb production which includes (i) use of animal is scientifically justified (ii) methods to be used which gives minimum pain to the animal1.Monoclonal antibody production (Past to Present)Mouse mAbsThis engineering science was introduced in 1975, which works on generation of mouse hybridomas by fusion of B cells, obtained from immunized mice, and myeloma cells. But mAbs produced by this method have many limitations and is not preferred due to high immunogenicity in humans and due to production of human anti-mouse antibody which top outs to their rapid clearance from unhurrieds body 3.Chimeric mAbs These are produced by gene manipulation method in which constant regions of mouse Abs are replaced by human Abs. Like mouse mAbs, chimerical mAbs also leads to make-up of human anti-mouse antibodies and leads to various immunogenicity in patients thus to make it potent in therapeutics further better understanding is required in their structure and function 3. valetized mAbsIn this method, complementary determining regions (CDRs) are transferred to human IgG from mouse mAb. There is only 5-10% non-human content in humanized mAbs in comparison to 30% in chimeric mAbs 3.Generation of mAbsImmunization of mice Screening of seraSpleen cell closing off Myeloma cells prep. Cell fusion(Tissue culture) Hybridoma screening Selecting cultures for cloning Mouse Feeder cells Cloning (limiting dilution) Clone isolation and expansion Cell freezing and recovery Supernatant production (from tissue culture media)mAbs purificatio n and testingFigure1. Flowchart showing steps for production of monoclonal antibodies by tissue culture method 1.Monoclonal Antibody Production Against various Diseases5C3 mAb against Tumor Growth and AngiogenesisIn this method, S100A4 was used for immunizing female Balb/cAnNHscl mice and mAbs were obtained from fused myeloma and spleen cells victimisation PEG-1500. Hybridomas were selected on HAT medium and further screened for it reaction with S100A4 by ELISA. Clones were selected which were corresponding to 5C3 mAb. Cell culture was scaled up in humid conditions (air 94% and 6% CO2) at 37C temperature 4. Supernatant ( blood serum free) from hybidomas was obtained and purified on column containing protein A with the help of AKTA purifier FPLC system and elutions containing 5C3 mAbs were concentrated and filtered in PBS centrifuge Amicon Ultra-15 which has low ski binding Ultracel membrane and then(prenominal) quantifying mAbs at 280nm 56.2-4F mAb against Oxytetracycline in sh rimpsOxytetracycline is used as medication feed in aquaculture 7, its overuse can lead to its assembling in aquaculture food and its consumption then leads to serious health problems in sea food lovers. To prevent consumers from its harmful effects mAb 2-4F, highly sensitive and specific, were produced for staining of OTC in aquaculture food animals by ELISA. Hybridomas were obtained by standard protocol, by immunizing the female BABL/c mice with OTC-BSA, hybridomas were cultured and supernatants from culture were screened for antibodies using iELISA and antibodies were cloned by limiting dilution method to obtain monoclones then in serum free media these moloclones were cultured in 500 ml spinner flask 67. Further mAbs were purified from this culture using protein G by affinity chromatography. The elute fractions were collected and its protein concentration was determined at 280nm spectometrically and mAb was filtered using cellulose acetate membrane (0.2 m) and kept at -20C unti l used 8.Human anti-human IL-21 monoclonal antibody.Interleukin-21 is a type I cytokine with four helical bundles that exerts effect on hematopoietic cells like NK cells, T and B lymphocytes. CD4+ T and NK T cells produce interleukin-2 cytokine, over expression of IL-2 lead to variety of autoimmune disorders. Genetically modified Kirin-Medarex mice were immunized with rhIL-21, immunogens were emulsified with P-adjuvant and CpG and recombinant mouse GM-CSF. Hybridomas obtained were cultured in IMDM containing 1x GlutMax, 1x Penicillin, 10% fetal clone serum and 10% Hybridoma Cloning Factor. Hybridomas were selected with IMDM in conjugation with HAT medium and cloning was carried out with 1x HT and distributed in 96 well Elisa plate and wells were examined microscopically for monoclonality and screened with phosphorylated-STAT3. Wells with positive results were distributed in 24 well cultures to obtained closeness 6105 cells/ml and then supernatant was collected and cells cryopreserv ed. Further media with human IgG was obtained and filtered through 0.2m membrane and from this filtered media antibody protein was purified by combing Protein G Sepharose Affinity Chromatography Size Exclusion Chromatography and absorbance was taken at 280nm and further its quality was accessed by size exclusion HPLC 9.mAbs L317, L363, L386 -galactosylceramideCD1d tortuousThe -galactosylceramide also known as KRN7000 is best studied ligand that binds to protein CD1d. KRN7000mCD1d complex is easily recognized by iNKT cells and leads to number of proinflammatory and immunoregulatory functions. To understand the mechanism of antigen presentation to CD1d by iNKT cell three monoclonal antibodies L317, L3363, and L386 were produced. Primary immunogen was prepared with protein obtained from strain H37Ra of Mycobacterium tuberculosis (PPD) and it was conjugated with the complex KRN7000CD1d. The complex KRN7000mCD1dPPD was studied by SDS-PAGE. Mice were eldest vaccinated with Mycobacterium bovis (BCG) then after 23 days mice were immunized with 5g KRN7000CD1dPPD complex in 11 PBS and Imject alum. At day 61 booster dose was given to mice, of the complex, with 7106 cells.Mice were then sacrificed and spleens dispersed PBS, cells were obtained and further washed with PBS and erythrocytes were lysed and cells were suspended in FBS/HEPES free DMEM 1011. The preparation was then mixed with myeloma cells and centrifuged and tubes with pellet were placed in water bath set at 40C and into this heated PEG was added followed by FBS/HEPES- free DMEM and then cells again centrifuged and re-suspended in DMEM. Hybridomas along with MRC-5 fibroblast feeder blast cells were plated in 96 well tissue culture plates. Supernatant from culture was screened and cloning of hybridomas carried out by limit dilution. Then 108 cells were inoculated in 2 liters roller bottles containing 500ml medium and OptiMAb supplement was added. MAbs were obtained by filtering of supernatant through protein G column chromatography 12.Stx2f-1, Stx2f-3, Stx2f-4 mAb against Shiga toxin, a gastrointestinal diseaseShiga Toxin 2 also designated as Stx2 is virulence causes gastrointestinal disease in humans world by food poisoning. It subtype Stx2f cannot be easily detected by immunological methods and thus three monoclonal antibodies specific to it were produced. Complete hybridoma media contains Iscoves modified DMM with NaHCO3 and 1 Glutamax, containing fetal calf serum (heat inactivated) 13. Female Balb/cJ mice were immunized with His-tagged Stx2f and hybridomas were obtained and screened for antibodies against Stx2f by ELISA and were further transferred to MPCM/HT/cHM media and diluted 500cells/ml and then the cells were grown in cHM media. Media containing antibody (400ml) was filtered through protein G column and elution were obtained in 0.1M glycine giving 5mg of purified antibody Stx2f 1415.Monoclonal antibody from EB66 Cell lines with heighten ADCC activityEB66 cell lines are deriv ed from embryonic stem cells of duck which can be genetically engineered and production of mAbs can be increased above 1g/L when grown in serum free media. EB66 have various other characteristic features like short doubling time, high cell density and unique metabolic profile with low accumulation of ammonium and lactate and low consumption of glutamine 16. Further, EB66 cell lines used for production of mAbs has reduced fucose content with enhanced ADCC activity. EB66 cell lines produce chimeric IgG1 anti-cancer mAb against antigen anti-X by nucleofection. EB66 clones when grown in Erlenmeyer flask with standard fed batch culture produces 1.28g/L of IgG1 of cell density with 36 millions cells/ml. Further by accumulation of monoclonal antibodies in supernatant culture no degradation was observed in antibody production assessed by HPLC, SDS-PAGE and western blot. When the supernatant was purified with Protein-A HPLC showed 98% mAbs as monomers. Glycosylation profile of monoclonal an tibodies was analyzed by MALDI-TOF-MS, enhanced activation of the monoclonal antibodies obtained from EB66 cell lines was analyzed by flow cytometry1617.FDA Approved mAbs in market 1819Infliximab Remicade TNFRituximab Rituxan, MabThera CD20Trastazumab Herceptin HER2Bevacizumab Avastin VEGFAdalimumab Humira TNFCetuximab Erbitux EGFRRanibizumab Lucentis VEGFPalivizumab Synagis RSVTositumomab Bexxar CD20Alemtuzumab Campath CD52Certolizumab pegol Cimiza TNFGemtuzumab ozogamicin Mylotarg CD33Muromonab-CD3 Orthoclone Okt3 CD3Efalizumab Raptciva CD11aAbciximab ReoPro GP IIb/IIIaBasiliximab Simulect CD25Eculizumab Soliris C5Natalizumab Tysabri a-4 integrinPanitumumab Vectibix EGFROmalizumab Xolair IgEDaclizumab Zenapax CD25Ibritumomab tiuxetan Zevalin CD20Recent advances in mAbs productionEngineered Monoclonal antibodiesAdvancement in mAb engenrreing has lead to transformation in this field which has lead to production of new drugs which as many useful characteristics like decreased immunog enicity, improved specifity along with stability and government agency 18. The replacements of murine as well as chimeric mAbs with full human mAbs are boon of this novel technology for example adalimumab, ranibizumab and cetrolizumab pegol. Adalimumab, the human mAb, is created by using bacteriophage display technology and now it is the top selling drug in the market. Cetrolizumab pegol has been engineered to increase its half-life by making changes in its Fab fragments 19. Ranibizumab which is derived from bevacizumab starchy AMD (age-related macular degeneration) and is considered as care indication standard. These new engineered mAbs have potential to compete with the drugs already in market and have bright future ahead 1920.Biosimalar Monoclonal antibodiesBiosimilars are the copies of drugs whose patient has expired and now these drugs can be produ--ced and manufactured by any company. But due to complex molecule used and then its approval from U.S makes it a complex process therefore most of the biotechnology companies are not in favor of production of biosimilars. Dr. Reddy in India has launched Reditux which is anti-CD20 monoclonal antibody and it is claimed, as the first biosimilar monoclonal antibody, by the company. In spite of approval of Reditux in India, it is thought that it would not have sufficient data that can fulfill the set standards of developed countries in terms of strict safety, efficacy and manufacturing standards181920.ConclusionMonoclonal antibodies are expanding rapidly in pharmaceutical industries with already hundreds of candidates are under increment and trials. Both cytotoxic and radiology methods are emerging to increase efficacy of the present therapeutic molecules. Moreover, advances have also been made to use mAbs in treatment of bacterial and viral infection. Biosimilars and bio-superiors are the next generation drugs which can be produced as most of the blockbuster monoclonal antibody are at verge to their discernibl e expiry. The future of the monoclonal antibodies in therapeutics is bright and continued discovery, research and development in this field can take it to the heights that have not been achieved before. purloinMonoclonal antibodies today have gained a breakthrough and are used in treatment of numbers of disease. Over 30% of the Engineered Monoclonal antibodies are under clinical trials. Moreover, different methods to generate human monoclonal antibodies are present today like generation of humanized and chimeric antibodies from genetic engineering of mouse antibodies, phage display method and transgenic mice development. Monoclonal antibodies are in great demand today and FDA has approved almost 22 mAbs till date and all these are commercially available in market. Biosilimars are also taking up the pace as most of the blockbuster mAbs are at verge of their patient expiry and Reditux developed by Dr. Reddy claimed as first biosimilar in India and is half the cost of Rituximab.