Biopharmaceutical Products Touqeer Ahmed Ph.D. Atta-ur-Rahman School of Applied Bioscience, National University of Sciences and Technology 23 rd September, 2014

Biopharmaceuticals Biopharmaceuticals are drugs produced using biotechnology. These could be proteins, nucleic acids, DNA, RNA used for therapeutic or for in-vivo diagnostic purposes. – Antibodies monoclonal (Various) – Blood factors (Factor VIII and Factor IX) – Thrombolytic agents (tissue plasminogen activator) – Hormones (insulin, glucagon, growth hormone, gonadotrophins) – Haematopoietic growth factors (Erythropoietin, colony stimulating factors) – Interferons (Interferons-α, -β, -γ) – Interleukin-based products (Interleukin-2) – Vaccines (Hepatitis B surface antigen) – Additional products (tumour necrosis factor) – Therapeutic enzymes

Antibodies Polyclonal antibodies Monolconal antibodies Bi-specific antibodies

Antibody Types Polyclonal antibodies (or antisera) are antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell Recombinant technology has led to the creation of “humanized” antibodies that overcome the immunologic problems previously observed following administration of mouse (murine) antibodies. Currently, several monoclonal antibodies are available in the United States for the treatment of cancer. Monoclonal Antibody Polyclonal Antibody

Antibodies Production

Polyclonal Antibodies

Polyclonal antibodies are heterogeneous, comprising a mixture of antibodies, each specific for one epitope. Advantages – Facilitates in the immune system by phagocytosis, and complement-mediated lysis of antigen in the in-vivo Antibody heterogeneity that increases immune protection in vivo, however, often reduces the efficacy of an antiserum for various in vitro uses. For most research, diagnostic, and therapeutic purposes, monoclonal antibodies, derived from a single clone, specific for a single epitope, are preferable.

Monoclonal Antibodies

Production of Monoclonal Antibodies Monoclonal antibodies are typically made by fusing myeloma cells (cancerous plasma cells) with the spleen cells from a mouse that has been immunized with the desired antigen Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium (HAT medium) in which only fused cells can grow is used.

Principle of the HAT selection Pathways of the nucleic acid synthesis – Salvage synthesis pathway (Needs HGPRT enzyme) Mutated in the myeloma cells – De novo synthesis pathway (blocked by the HAT medium) HAT = hypoxanthine, aminopterin, and thymidine The combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor, with hyproxanthine and thymidine, which are intermediates in DNA synthesis, provides a form of artificial selection for cells containing functioning hypoxanthine-guanine phosphoribosyltransferase (HGPRT) HAT Selection HGPRT= hypoxanthine-guanine phosphoribosyltransferase

Purification of the Monoclonal Antibodies Media from the hybridomas (having antibodies) – Contaminants (growth factors, hormones and cytokines) Ascites fluid (having antibodies) – Contaminants (antibodies, proteases, nucleases, nucleic acids, viruses, bacterial endotoxins and cytokines) Purification approaches Centrifugation and then filtration using 0.45 um filter (to remove large particles and debris) Ion exchange and size exclusion chromatography Protein A/G affinity chromatography Affinity purification using antigen Antibody precipitations (using sodium sulphate or ammonium sulphate) as Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations Gel electrophoresis to make sure the purity

Naming Monoclonal Antibodies