Challenges in Manufacturing of Biopharmaceutical. Amulya K Panda National Institute of Immunology New Delhi

Transcription

1 Challenges in Manufacturing of Biopharmaceutical Amulya K Panda National Institute of Immunology New Delhi Theatre, Indian Habitat Centre, New Delhi July 30 31, 2013

2 Outline of talk 1. Bioprocessing of Recombinant Proteins 1. Challenges Using E. coli 2. Yeast and CHO cell Platform 4. Innovations in Biomanufacturing

3 Important Model Bio-Processes 1. Penicillin from P. chrysogenum 2. Conversion of Glucose to HFCS 3. Insulin from recombinant E.coli 4. Shikonin from Lithospermum erythrorizon 5. Hepatitis B surface antigen from Yeast 6. EPO/ HuMab from CHO/NSO cell lines 7. PHB from Alcaligenus/ E. coli?

4 History of Penicillin Production Year Discovery Penicillin Yield (g/l) >60 70 ~ Cost $/Kg 15,

5 Penicillin 4 Amino acids 25 US $/Kg Insulin 51 Amino acids US $ 300/ Kg Assume Insulin is 100 times more complex than Penicillin Cost of production should be US$ 30/g Hepatitis B 227 Amino acids US $ 1300/Kg Assume HBsAg is 1000 times complex than Penicillin Cost of production should be US$ 1000/g That is US$ 1 /mg = US$ 0.02/dose (20 g) Rs 1.00/20 g (one single dose)

6 Reduction in Production Cost 1. High Volumetric Productivity gm/l/hr should be high,(high cell density fermentation, perfusion culture, continuous culture 2. High Throughput Purification High recovery of bioactive protein, use of new matrix/novel column/purification strategy/refolding operation 3. Improved formulation for stability and high half-life during circulation stabilizers, PEGylation,protein engineering cont. release formulation

7 Recombinant Proteins from E.coli Insulin IGF/ IL-2 Streptokinase FactorX/ TPA GCSF GM colony stimulating factor IFN- /IFN- Human growth hormone Bovine growth hormone Calcitonin/Bone morphogenic protein Exantide/PTH Objective : To maximize volumetric productivity To purify maximum of expressed protein To formulate protein into a stable form

8 Biosimilar Product Manufacturing 1. Purity 2. Efficacy 3. Safety 4. Similarity 5. Affordability - high yielding strain - high cell density fermentation - high throughput recovery/purification - novel formulation and stability

9 Biological Engineering Cascade for Biosimilar Manufacturing

10 Gene expression in E. coli Transcription Translation DNA Copy no: Promotor mrna Protein Folding Compartmentation? Secretion? Replication Decay Decay CO 2 HAc Nutrients - ph - Temp - O 2

11 Parameters Influencing the Productivity of Recombinant protein in E. coli 1. Parameters relating to DNA copy no., stability, replication 2. Parameters relating to protein synthesis promoters, terminators, mrna stability, transcription and translation efficiency 3. Parameters relating to protein toxicity, fusion protein, inclusion body, proteolysis 4. Parameters relating to fermentation bioreactor operation, growth-product relationship, induction strategy, change in cellular metabolism 5. Parameters relating to downstream processing cell harvest, separation, purification, refolding, bioactivity and containment

12 Cell Engineering for High Expression of Protein 1. Host genotype and physiology -protease deficient strain, low acetate secretor 2. Translation initiation region multiple promoter, tandem repeat of gene, polycistronic vector 3. Introduction of chaperone - Dsb/Dsc, PDI 4. Manipulation of transport system -signal sequence

13 High Cell Density Fermentation Principle : Allow the cells to grow in log phase for considerable time period by proper nutrient supply and controlled environment grams dry cell weight/l of E. coli can be achieved by use of fed-batch fermentation Advantages: Increased volumetric productivity Improved cell separation Less waste disposal problem

14 High cell density growth of E. coli using different feeding strategies

15 Problems of scaling up process from shaker flask to large scale reactor 1. Number of generation is more (need special care to maintain plasmid stability). 2. How to maintain specific protein yield at high cell concentration? 3. How to recover large fraction of bioactive protein from inclusion bodies

16 Strategy for E. coli growth and recombinant protein production Supply glucose along with optimal amount (C/N : 1 : 0.75)of complex nutrients such as yeast extract at predetermined growth rate ( ~0.15 to 0.25). Adavantages: (1) Reduce metabolic burden as glucose is mostly used for energy generation whereas nitrogen source provides building blocks for the cell, promotes cell growth (2) Enhance acetate assimilation, better buffering of the medium, provide amino acids for high level synthesis of protein. Maintain specific yield of protein

17 Glucose and Acetic acid conc.(g/l) Cell O.D. at 600nm T o S a v e t h i s t e m p l a t e, C h o o s e F i l e : T e m p l a t e : T e m p l a t e S a v e. hgh conc. (g/l) 200 HIgh cell density feremntation of E.coli for production of recombinant human growth hormone hgh Conc.(g/L) Acetic acid(g/l) Glucose conc.(g/l) Cell O.D. at 600nm Time (hrs)

18 hgh production during fed-batch fermentation Cell conc. at IPTG induction (OD 600nm) Final cell Conc. (OD at 600nm) hgh Conc. (gl -1 ) Specific r-gh yield (mgg -1 of cells) Shake flask 2 OD 55 mg/l (6 gm dcw) (64 gm dcw)

19 Recombinant Protein Expression Using High Cell Density Fermentation Protein Cell Conc. (gl -1 ) Ferment Time (h) Protein Conc. (gl -1 ) HGH Gal pro Insulin HGH fusion IFN Insulin Bio Adh Protein IGF hgh PHB Leptin

20 Recovery to Fermentation cost 0.16 for Ethanol 1.0 for Penicillin 2.0 for Enzymes 3-5 for therapeutic proteins from E.coli Recovery cost of bioactive protein from inclusion bodies accounts > % of total production cost For TPA around % For growth hormone 50 % Aim High throughput recovery a. Protein folding at high concentrations ( > 200 mg/l) b. High recovery from IB (>25 %)

21 Protein Purification from Inclusion Body Cells IB Denatured protein Refolded protein Pure refolded protein French press /sonication) Solubilization in 8M urea/gnhcl Refolding by dilution Chromatography Problems: Low yield of pure protein (15-25%) Refolding at low concentration (10-50µg/ml) Urea and water constitute 50% of the raw material cost in insulin production plant.

22 Steps in refolding of inclusion body protein 1. Isolation of IB from Cell 2. Solubilization in Aggregates 3. Refolding of solubilized protein 4. Purification of refolded protein Can we improve the recovery of bioactive protein by optimization/novel methods in all these steps

23 Mild solubilization of inclusion body proteins/ Human growth hormone 1. Solubilization by ph shock (alkaline/acidic ph) 2. Solubilization by detergent (CTAB/NLS) 3. Solubilization by Low urea/l-arginine 4. Solubilization by high pressure (2 Kbar) 5. Solubilization by organic solvents Many of these protect existing native-like secondary structure of the inclusion body protein aggregates

24 Inclusion Bodies Soft inclusion bodies (Asparaginase) Tough inclusion bodies (human growth hormone) 1. Smaller in size (< 200 nm), soft and relatively less dense 2. Need low concentration of chaotropes for solubilization 3. More susceptible to denaturant and proteases 4. Proteins have secondary structure and residual bioactivity 5. Less amyloidogenic than the classical IBs 1. Larger in size ( nm), tough, dense 2. Need high concentration of chaotropes for solubilization 3. Less susceptible to denaturant and protease resistant 4. Proteins have secondary structure in IBs 5. Highly amyloidogenic in nature

25 Improved Refolding of Inclusion Body Proteins 1. Purify inclusion body to homogeneity lyses cells carefully, purify IB by detergent washing /ultracentrifuge (If IB are pure, no need of any Tag/ less chromatography steps) 2. Determine whether inclusion bodies are soft/ tough ( concentration dependent urea denaturation/ take care of pure IB ) 3. Solubilize IB protein using mild solubilizing agent (high ph/pressure/ propanol/β-mercaptoethanol/2-3 M urea or arginine) 4. Refold using pulsatile renaturation with optimal refolding buffer ( refolding at high protein concentration ( mg/l) 5. Purify and lyophilize (use radial flow to process huge volumes of diluted protein, lyophilize with stabilizers)

26 Steps Purification of r-hgh from IB of E. coli Total protein in mg. Step yield Overall Yield (Crude protein+ib) 2000 mg 100% 100% Pure Inclusion bodies 220 mg 100% 100% Solubilization 175 mg 79.5% 79.5% Refolding 161 mg 92% 73.2% Ion exchange chromatography 120 mg 68.5% 54.5% Gel filtration chromatography 100 mg 83.3% 45.4%

27 One of the strongest eukaryotic promoters known Gene product alcohol oxidase 1 Initial step in methanol metabolism CH 3 OH O 2 H 2 O 2 CAT O 2 + H 2 O HCHO CO 2 Tightly regulated Repressed on Glucose Glycerol Ethanol Highly induced on methanol 30 % of soluble protein Biomass Protein Expression in Pichia using AOX1 Promotor

28 HSA Fusion proteins expression in Pichia pastoris HSA Fusion Protein Titer secreted (g/l) HSA 18 Transferrin 11 HSA-IFNα2a 16.5 HSA-GCSF 18.0 GCSF-HSA 20.0 Scaffold 1-HSA 15.0 Scaffold2-HSA 10.0 Scaffold3-HSA 9.0

29 Steps in glycoprotein production

30 Growth and productivity of two GS-CHO cell lines making the same chimeric antibody: comparison of a process from 1990 to 2005

31 What is heterogeneity from a regulatory perspective? ICH Q6B Drug Substance/Drug Product Desired product Heterogeneity (product-related Variants) Expacted product (cdna) + Product-related substance Product-related impurities Process-related impurities Contaminants Purity: highly method dependent Combination of analytical approaches Requested both for the DS and the DP the presence of some heterogeneity clearly accepted. Impurities: they can be process and product related. Contaminants: material not intended to be part of the manufacturing process

32 Ideal Expression levels for Biosimilar Protein Production 1. E. coli gm/l, > 50 % recovery (1 day cycle) 2. Pichia pastoris gm/l (secreted product), > 70 % recovery 3. CHO/ HEK/NSO 6-8 gm/l (secreted) > 70 % recovery

33 Insulin Requirement 500mg/person/year (Type I) World Diabetic population and requirement million million million Indian Diabetic Population and requirement million million million Assume 10% Type I Diabetic Requirement for India is 5 Ton (2020)

34 Innovation in Biosimilar Product (Insulin) 1. Different expression system (E. coli / Lactobacillus / yeast /CHO / fungus) 2. Different version of insulin (normal/ long acting/first acting/peg-insulin) 3. Novel way of purification and refolding 4. Novel way of delivering insulin (oral/mucosal/single dose) Noble prizes have been awarded four times on different aspects of insulin

35 Innovations in Bio Manufacturing 1. Use E. coli that do not produce endotoxin 2. Use E. coli that can produce glycosylated protein 3. Phage mediated E. coli expression of soluble protein 4. Engineer yeast for methanol free induction 5. Engineer yeast for controlled glycosylation 6. Perfusion strategy for high density growth of CHO cells 7. Homogeneity of glycoform during high cell growth 8. Glycoprotein expression using human cell lines 9. Expression of conjugated protein for long circulation 10. Protein purification using crystallization

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37 Characteristics of E. coli 1. Mol. Formulae : CH 1.77 O 0.49 N 0.24 Components : (~ 20% solid) 55% protein, 20% RNA, 5% DNA, 9% Lipid, 6% Polysaccharides. 2. Cell Volume V= 0.4X 2 ( = 0.05 to 1.3) = 0.69 V=0.6 m 3 1 L = m 3 No of cells /L = 1.5X10 15 E. coli, 150 g/10 9 cells 225 g / L (500 OD at 600nm) 3. Growth behaviour Facultattive O 2 requirement m mole 2 /g.cells/hr Product Extracellular, endotoxin, cell recovery

38 Glycosylation of recombinant protein expressed in two commonly used mammalian cell lines HEK293 and CHO-S Large differences in the glycosylation of recombinant proteins expressed under standard culture conditions HEK and CHO cells. Different apparent molecular weights on SDS-PAGE Different isoform pattern on isoelectricfocusing. Different composition and complexity of the N-linked glycostructures. Differences in the glycosylation was detected between the two HEK cell lines for some of the proteins. Change in culture/transfection conditions showed no or only minor effect on the glycosylation

39 Novel Method of Protein Recovery from Inclusion Body Isolate pure inclusion body Detergent Washing / Sucrose Gradient Centrifugation Solubilize IB Proteins Low Conc. Denaturants Refold/Buffer exchange Purify and lyophilize

40 Refolding of solubilized hgh 1. IB of hgh solubilized in - Mercaptoethanol X Pulsatile dilution of - Me in buffer containing sucrose 3. Purification by radial as well as axial column chromatography Pulses of solubilized protein added in to large volumes of buffer and allowed to refold. Refolded protein never co-aggregate with refolded protein, thus large quantity of the protein can be refolded in same volumes

41 Conclusion Biosimilarirty start at the CMC level. A product is not born biosimilar, it is made biosimilar by adjusting the manufacturing/purifaction processes. Biosimilar must be equivalent pharmacokinetically and pharmacodynamically in adequately powered studies. Comparable efficacy and safety must be demonstrated in head-to-head comparison with narrow pre-specified equivalence criteria. Clinical studies should be long enough to assess immunogenicity using validated assays

42 Comparable quality, safety and efficacy is the overarching theme in development of biosimilar biopharmaceuticals Quality cannot be tested into a product, it has to be built in by design International guideline ICH Q8 Quality and comparability considered during all stages of development Manufacturing innovation and state of the art technology. Serum and antibiotic free cell banks and manufacturing. Less protein oxidation, silicon oil, particles, aggregates, extended self live

43 Formulation and stability studies Extended stability Use of stabilizer (s) and its concentration Product quality in formulated condition Bioactivity/immunogenicity of the formulated product

44 A Biological Reaction B + C + D Concentration : g/l Conversion : How much A is consumed Yield : Amount of cell/product per unit amount of A Specific Yield : Amount of product/per cell Productivity : gm/l/hr of the product

45 GLUCOSE PEPTONE YEAST EXTRACT ACTIVE CELL GAPDH ACETATE BIOMASS

46 1. If the protein is refolded from Inclusion bodies (a) show details of refolding process (b) show comparative bioactivity at different doses (c) show stability data (refolded protein is stable, soluble and does not show aggregating behavior 2. If the protein is glycoprotein (a) Complete glycan profile (b) Specific carbohydrate position if any ( c ) Bioactivity at different dose

47 Implications Recombinant proteins expressed in mammalian expression system are not identical to the native protein. Protein expressed in different mammalian cell lines have different glycosylation, thus are not identical. When comparing biochemical /biophysical properties and biological activity of recombinant proteins the expression system used for the production need to be consider, even when the protein was produced in different mammalian expression systems.

48 Engineering Protein Drugs (Formulation) 1. Reduce immunogenicity ( human monoclonal antibody) 2. Increase bioavailability (insulin) 3. Increases circulation time (pegylation) 4. Conjugated protein 5. Polymer particle/ liposomal delivery system

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