Expression of Recombinant Biomedical Products from Mammalian Cell.pptx

Recombinant Biomedical Products from Mammalian Cell

1. Expression of Recombinant Biomedical Products from Mammalian Cell Lines

2. Introduction The ability to produce mammalian proteins in recombinant systems has had a profound impact in many areas of basic and applied research, as well as the biotech sector. Basic research- functional analysis, structure determination, interaction study Applied research- clinical trials Biotech industry- protein therapeutics commercial manufacturing

3. Introduction The production of recombinant proteins for biopharmaceutical use is a multi-billion dollar industry, with global sales over US$120 billion per year (2011) and US$150 billion (2015). Monoclonal antibodies are the largest class of biologic drug in term of sales. While E. coli , yeast and insect cell lines are still the preferred platforms for many protein expression groups, mammalian proteins often require mammalian cells for optimum yields and activity. The bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins.

4. US sales ($ billions) of top nine classes of biologic drugs in 2011

5. Key Requirements Expression system Vector backbone Gene delivery and localization Selection system Downstream processing

6. Expression System CHO (Chinese Hamster Ovary) HEK293 ( Human Embryonic Kidney) NS0 (Non secreting murine myeloma  cells) CHO cell-based systems remain by far the most common mammalian cell line in use; 84% are produced in CHO systems.

7. CHO Cell Line The workhorse of mammalian protein production (especially at industrial scale) is the CHO cell line, isolated by Theodore Puck in the late 1950’s. Since the commercial introduction of human tissue plasminogen activator (tPA) as the first recombinant therapeutic protein produced from mammalian cells, the annual global revenue of products from CHO cells has increased to more than US$100 billion and continues to grow. Strength adaptability to grow at high densities in suspension cultures ease of adaptation to chemically defined and serum free conditions ideal from regulatory standpoint since most human pathogens do not replicate Relatively high yield in heterologous expression (2-6 gm/l) Genomic variability results in mutant lines that provides selection strategies

8. HEK 293 Developed during 1970a, the 293 cell line was the first human line to be transformed using sheared adenovirus DNA fragments of the Ad5 serotype Since its development, the 293 cell line has become one of the most commonly used human cell lines for protein production Strength Proteins produced in HEK cells are a closer match to naturally occurring human proteins in terms of post translational modification and function Diverse variants exist that support suspension growth (293N3S) Growth under serum free condition (293S) Transient gene expression by episomal replication of plasmids (293 T and 293 E)

9. Mammalian Expression Vectors Plasmid-based vectors Viral vectors

10. Plasmid-based Vector

11. Genetic Elements of Mammalian Expression Vectors Origin of replication (Mammalian) Promoter Eukaryotic ribosome binding site (Kozak sequence) Polyadenylation signal Transcription termination signal Selectable marker gene in mammalian cell Prokaryotic origin of replication Prokaryotic selectable marker Mammalian expression vector backbone Prokaryotic cloning vector backbone Shuttle vector backbone

12. Genetic Elements of Mammalian Expression Vectors

13. Viral Vectors for Mammalian Expression Herpes simplex virus Epstein-Barr virus Simian virus 40 Adenovirus Adeno-associated virus Vaccinia virus Corona virus Polio virus Lentivirus Retrovirus

14. Viral Delivery Lentiviral vectors (LVs) derived from human immunodeficiency virus type-1 (HIV-1) have a long history of efficient gene delivery in gene therapy applications. The ‘Daedalus’ system has shown promising results for the rapid generation of stable producer lines in HEK 293 Freestyle cells (Invitrogen), with yields of 20–100 mg/L reported for a variety of secreted proteins without the need for clonal selection. LVs have also been used in the CHO platform where certain clones were shown to produce as much as 200 mg/L of the human tumor necrosis factor receptor-Fc fusion protein. One of the drawbacks of the LV system is the modest packaging size of the lentivirus capsid (10 kilobases), which may limit the maximum size of the recombinant protein being expressed .

15. Protein therapeutics (including monoclonal antibodies [ mAbs ], peptides and recombinant proteins) represent the largest group of new products in development by the biopharmaceutical industry. mAbs continue to reign supreme, although cellular and gene therapies are slowly starting to gather momentum. Burgeoning growth in biosimilars may threaten future brand monopolies for mAbs and other biologics Overall, new approvals followed relatively predictable lines, with cancer representing the single most common indication (33 products). Other common indications included various inflammation-related conditions (24 products), hemophilia (16 products) and diabetes (15 products).

16. The cumulative sales of biopharmaceuticals over 2014–2017 reached $651 billion, whereas total sales for 2017 alone reached $188 billion The mAb Humira (adalimumab) has been by far the single most lucrative product each year during the survey period, having generated global sales just short of $19 billion in 2017 and $62.6 billion cumulatively between 2014 and 2017

17. Biopharmaceuticals are produced in a wide variety of platforms, including non-mammalian expression systems (bacterial, yeast, plant and insect) and mammalian expression systems. Importantly, the most appropriate expression system depends on the particular protein to be expressed. Mammalian expression systems are generally the preferred platform for manufacturing biopharmaceuticals, as these cell lines are able to produce large, complex proteins with post-translational modifications similar to those produced in humans Moreover, in the case of mammalian cell lines, and animal cell lines in general, most proteins can be secreted rather than requiring cell lysis to extract with subsequent protein refolding.

18. Humira (Adalimumab) Adalimumab , sold under the brand name  Humira , among others is a  monoclonal antibody  Humira stands for " hu man  m onoclonal antibody i n r heumatoid a rthritis“  it is indicated for the treatment of: plaque psoriasis (a disease causing red, scaly patches on the skin) psoriatic arthritis (a disease causing red, scaly patches on the skin with inflammation of the joints) rheumatoid arthritis (a disease causing inflammation of the joints) axial spondyloarthritis (inflammation of the spine causing back pain), including ankylosing spondylitis and when X-ray does not show disease but there are clear signs of inflammation polyarticular juvenile idiopathic arthritis and active enthesitis -related arthritis (both rare diseases causing inflammation in the joints) Crohn's disease (a disease causing inflammation of the gut) ulcerative colitis (a disease causing inflammation and ulcers in the lining of the gut) hidradenitis suppurativa (acne inversa ), a long-term skin disease that causes lumps, abscesses (collections of pus) and scarring on the skin non-infectious uveitis (inflammation of the layer beneath the white of the eyeball) chronic cases of aggressive progressive pulmonary and bone sarcoidosis

19. Vectors of Heterologous Expression in Animal Cells Expression Transient Stable Expression Type Vector Class Promoter Host System Transient Plasmid SV40 promoter Cytomegalovirus promoter Elongation factor 1 promoter HEK-293 COS BHK Viral Semliki Forest Virus promoter Vaccinia virus late promoter Adenovirus promoter Stable Linearized DNA vector CHO Viral Retroviral promoter with specific region of homology for ribosomal DNA