Saturday, September 21, 2019
Monoclonal Antibody Production using Animal Cell Culture
Monoclonal Antibody Production using Animal Cell Culture Introduction Monoclonal antibodies, in biomedical research, are used as reagents in diagnosis and treatment of diseases like cancer and infections . It has been almost century their introduction, mAbs are still produced from splenocytes fused to myeloma cells . 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 . For production of desired monoclonal antibodies, the cells should grow in one of the two ways: injecting the peritoneal cavity of mouse (known as in vivo method or mice ascites method) or by in vitro method (Tissue culture method). Further mouse ascites fluid or supernatant of tissue culture is processed and monoclonal antibody of desired concentration and purity is obtained (figure1) . Mice ascites method is preferred as it is familiar, properly understood and extensively used in laboratories in comparison to tis sue culture method which is time consuming, expensive and laborious and ails to give required amount of antibodies. Presently, twenty two monoclonal antibodies for transplantation, oncology, infectious, cardiovascular and chronic inflammatory disease have been approved by FDA . Strict guidelines has been setup 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 animal. Monoclonal antibody production (Past to Present) Mouse mAbs This technology 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 leads to their rapid clearance from patientÃ¢â¬â¢s body . Chimeric mAbs These are produced by gene manipulation method in which constant regions of mouse Abs are replaced by human Abs. Like mouse mAbs, chimeric mAbs also leads to formation 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 . Humanized mAbs In 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 . Generation of mAbs Immunization of mice Screening of sera Spleen cell isolation 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 purification and testing Figure1. Flowchart showing steps for production of monoclonal antibodies by tissue culture method . Monoclonal Antibody Production Against various Diseases 5C3 mAb against Tumor Growth and Angiogenesis In this method, S100A4 was used for immunizing female Balb/cAnNHscl mice and mAbs were obtained from fused myeloma and spleen cells using 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 37Ã °C temperature . Supernatant (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 binding Ultracel membrane and then quantifying mAbs at 280nm . 2-4F mAb against Oxytetracycline in shrimps Oxytetracycline is used as medication feed in aquaculture , its overuse can lead to its accumulation 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 detection 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 . 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 Ã µm) and kept at -20Ã °C until used . 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 density 6105 cells/ml and then supernatant was collected and cells cryopreserved. Further media with human IgG was obtained and filter ed through 0.2Ã µm 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 . mAbs L317, L363, L386 Ã ±-galactosylceramide:CD1d complex The Ã ±-galactosylceramide also known as KRN7000 is best studied ligand that binds to protein CD1d. KRN7000:mCD1d 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 KRN7000:CD1d. The complex KRN7000:mCD1d:PPD was studied by SDS-PAGE. Mice were first vaccinated with Mycobacterium bovis (BCG) then after 23 days mice were immunized with 5Ã µg KRN7000:CD1d:PPD complex in 1:1 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 . The preparation was then mixed with myeloma cells and centrifuged and tubes with pellet were placed in water bath set at 40Ã °C 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 . Stx2f-1, Stx2f-3, Stx2f-4 mAb against Shiga toxin, a gastrointestinal disease Shiga Toxin 2 also designated as Stx2 is virulence causes gastrointestinal disease in humansÃ¢â¬â¢ 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 IscoveÃ¢â¬â¢s modified DMM with NaHCO3 and 1 Glutamax, containing fetal calf serum (heat inactivated) . 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 . Monoclonal antibody from EB66 Cell lines with enhanced ADCC activity EB66 cell lines are derived 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 . 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 antibodies was analyzed by MALDI-TOF-MS, enhanced activation of the monoclonal antibodies obtained from EB66 cell lines was analyzed by flow cytometry. FDA Approved mAbs in market  Infliximab RemicadeÃ ® TNF Rituximab RituxanÃ ®, MabTheraÃ ® CD20 Trastazumab HerceptinÃ ® HER2 Bevacizumab AvastinÃ ® VEGF Adalimumab HumiraÃ ® TNF Cetuximab ErbituxÃ ® EGFR Ranibizumab LucentisÃ ® VEGF Palivizumab SynagisÃ ® RSV Tositumomab BexxarÃ ® CD20 Alemtuzumab CampathÃ ® CD52 Certolizumab pegol CimizaÃ ® TNF Gemtuzumab ozogamicin MylotargÃ ® CD33 Muromonab-CD3 Orthoclone Okt3Ã ® CD3 Efalizumab RaptcivaÃ ® CD11a Abciximab ReoProÃ ® GP IIb/IIIa Basiliximab SimulectÃ ® CD25 Eculizumab SolirisÃ ® C5 Natalizumab TysabriÃ ® a-4 integrin Panitumumab VectibixÃ ® EGFR Omalizumab XolairÃ ® IgE Daclizumab ZenapaxÃ ® CD25 Ibritumomab tiuxetan ZevalinÃ ® CD20 Recent advances in mAbs production Engineered Monoclonal antibodies Advancement 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 immunogenicity, improved specifity along with stability and potency . 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 phage 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 . Ranibizumab which is derived from bevacizumab wet 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 . Biosimalar Monoclonal antibodies Biosimilars 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 RedituxÃ ® which is anti-CD20 monoclonal antibody and it is claimed, as the first biosimilar monoclonal antibody, by the company. In spite of approval of RedituxÃ ® 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 standards. Conclusion Monoclonal antibodies are expanding rapidly in pharmaceutical industries with already hundreds of candidates are under development 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 patent 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. Abstract Monoclonal 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 RedituxÃ ® developed by Dr. Reddy claimed as first biosimilar in India and is half the cost of RituximabÃ ®.