Biotechnology Drugs

1. Biotechnology Drugs • Part I Basic concepts • Part II Process: Quality & Safety issues • Part III A checklist for inspection

2. Synthetic Drugs Small molecules Easy synthesis Aspirin Oligopeptides Hemi-synthesis Need of active stereo-isomers Steroids Anticancer Antimetabolites cyclosporin Extraction Biologicals Complex & rare molecules Animal source possible Insulin Heparins Human source necessary HGH Coag. Factors Albumin Why Are Biotechnology Products Different From Simple Organic Chemicals?

3. Biotech Drugs Better yield & better safety • Insulin • HGH Modified molecules Extraction impossible • Erythropoietin • Interferon • Interleukin • Growth factors

4. Biotech Products Produced by purification Starting material variable Many in-process tests End-product tests complex Process is product-specific Batch sizes small (g or kg of finished product) Conventional drugs Produced by formulation Starting material defined Few in-process tests End-product tests simple Process is product type specific (generalizations possible) Manufacturing Processes Are Different

5. Biotechnology Drugs • Use of micro-organisms (procaryotic or eucaryotic) genetically modified for production of complex molecules • After purification, the products are used in human or animal therapeutics

6. Medicinal Products Derived From Recombinant DNA • Insertion of naturally occurring or synthetic nucleotide sequence into a vector • Introduced into a suitable host organism to ensure the efficient expression of the desired gene product

7. Five Features For a Biotechnology Drug • Expression system of the gene • Production system compatible with the microorganism • Purification system • Nature of the active product • Pharmaceutical formulation and presentation

8. Five Features For a Biotechnology Drug1. Expression System: Vector + Host Identify, isolate and clone the gene coding for the desired protein Construct a vector containing: • The gene • The expression controls (promoter, secretion signal…) Insert the vector into the selected micro-organism • Escherichia coli • Saccharomyces cerevisiae • Mammalian cells • Genetically modified plants

9. Expression System: Vector Construction + Host

10. Five Features For a Biotechnology Drug2. The Production System Purpose • Optimize survival conditions for the genetically modified microorganism • So that it produces the desired protein • With acceptable yield Materials & Methods • Selection of cell culture medium • Selection of culture conditions • Selection of culture equipments:fermentor, cytocultor

11. Five Features For a Biotechnology Drug3. The Purification System Purpose • extract protein from a complex growth medium • Achieve close to 100% purity • Without altering the protein Materials & Methods • Sequence of purification steps • Filtration/ultrafiltration • Precipitation/resolubilization • Chromatography (ion-exchange, affinity, etc.)

12. Five Features For a Biotechnology Drug4. Nature of Active Product Proteins • Chains of amino-acids • Sequence of amino-acids encoded by genes • Folded into 3-D conformation • To obtain biological activity • Host-dependent post-translational modifications (sugars…)

13. Five Features For a Biotechnology Drug5. Pharmaceutical Formulation and Presentation Purpose • Maintain biological activity • By maintaining active protein conformation in solution Materials & Methods • Stabilization (albumin, glycerol) • Storage at low temperature

14. Biotechnology Drugs • Part I Basic concepts • Part II Process: Quality & Safety issues • Part III A checklist for inspection

15. Process Flow-Chart Fermentation- culture Harvest Starting material Purification Pharmaceutical finishing

16. A Cell Bank • A collection of ampoules of uniform composition stored under defined conditions, each containing an aliquot of a single pool of cells

17. The Master Cell Bank (MCB) • Generally derived from the selected cell clone containing the expression construct. • The MCB is used to derive all working cell banks.

18. The Manufacturer Working Cell Bank (MWCB) • Derived by expansion of one or more ampoules of the MCB under defined culture conditions • The working cell bank is used for the production of the batches.

19. The Late Expanded Cell Bank (LECB) • Obtained in multiplying the cells used for the production of the recombinant protein, several passage after the passage of production. • It is used to reveal a potential low viral contamination and to study genetic stability of the transgene.

20. Cell Bank System Genetic transformation Parental cell line Master cell bank (MCB) expand Starting material Manufacturer working cell bank (MWCB) expand Production substrate expand Late expanded cell bank (LECB)

21. Cell Bank System

22. Process Flow-Chart Fermentation- culture • Various expansion systems • (fermentor, roller bottle, fermenting flasks, • Cell culture systems, airlift, hollow fiber • Various culture media • (totally synthetic or containing components • of animal origin) Starting material

23. Process Flow-Chart Fermentation- culture Harvest Starting material • Discontinuous production system • (from fermentor) • Continuous production system • (several harvests of culture supernatant)

24. Process Flow-Chart Fermentation- culture Harvest Starting material Purification • Various extraction strategies • (from supernatant or cell disruption) • Various purification methods • (according to potential contaminants)

25. Purification: Potential Contaminants • What contaminants may be encountered? • What is the source of the contamination? • How hazardous are they? • How can they be removed? • How can we ensure their removal?

26. Process-related impurities Cell substrate derived Cell culture derived Downstream derived Product-related impurities Truncated forms Other modified forms aggregates Purification: Potential Contaminants

27. Purification: Potential Contaminants Microbiological contaminants:adventitious agents • Prevent in the original cells or in the master cell bank • Adventitious viruses introduced during production

28. Purification: Potential Contaminants Hazards: • Toxicity • Heavy metals, cyanogen bromide, antibiotics, organic solvents… • Altered pharmacological activity • Aggregates, breakdown products • Immunogenicity • Oncogenicity • Infectious diseases • Mycoplasmas, yeasts, viruses

29. Purification: Contaminant Removal A sequence of several methods: • Precipitation • Filtration • Liquid chromatography based on different physical or chemical principles • Affinity, hydrophobicity, molecular weight, electrical charge…

30. Biotechnology Drugs • Part I Basic concepts • Part II Process: Quality & Safety issues • Part III A checklist for inspection

31. Purification System Should Be: • Appropriate • Justified • Validated

32. Process Flow-Chart Fermentation- culture Harvest Starting material Purification Pharmaceutical finishing • Stabilization • (according to protein and delivery mode) • Storge

33. A Checklist For Inspection • Focus on changes in the manufacturing process • Do not classify a priori a change in the process as minor or major • Consider the potential consequences on the drug product in terms of : • Quality • Safety • Efficacy

34. Changes In the Manufacturing Process Main reasons for introducing changes • Improvement of product quality • Increase of production yield • Global productivity • Cost savings • Production scale-up • New production sites

35. A Checklist For Inspection 1.Cell bank system 2.Fermentation/culture process 3.Purification process 4.Drug substance 5.Formulating and filling 6.Drug product

36. Issues For Inspection:1.Cell Bank System • GMP compliant plant design • Documentation of cell origin • Setup conditions for the (Master), (working) and (late expanded) cell banks: • Documentation of viral safety • Documentation of expansion conditions • Storage conditions • Measures taken against contamination

37. Issues For Inspection:2.Fermentation/Culture Process • Starting material (cells) • New supplier • Specifications • Additions/substitution of new material • Cell culture conditions • pH, oxygen, temperature, time, % of reagents, formulation of culture media… • Scale of fermentation/cell culture mode • Equipment • Change of additional fermentation site or facility

38. Issues For Inspection:3.Purification Process • Column/resin change • Size of column, supplier, cleaning conditions, storage conditions • Reagents • New supplier, specifications, replacement of raw material • Purification protocols • Addition/substitution/elimination of specific steps • Scale of the downstream processing • equipment

39. Issues For Inspection:4.Drug Substance(active principle) • Batch definition • Storage conditions • Pooling strategy

40. Issues For Inspection:5.Formulating and Filling • Excipient • Equipment • Manufacturing process • Scale • Change of site/facility • Storage & shipping conditions

41. Issues For Inspection:6.Drug Product (final product) Control of : • Identity • Purity • Activity • Stability • General safety • Sterility • pyrogenicity

42. Issues For Inspection:Summary • Clean starting material • Well-characterized cell-line • (cell bank) system • Validated production & purification processed • (In-process) controls • Specific final products testing • Compliance to GMPs

43. Conclusion The growing number of biotechnology products requires highly skilled professionals to inspect complex and changing production processes