bt3021 2015 lecs 1 3 introduction to dsp
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Same as Title. Guhan sir's slides.TRANSCRIPT
BT 3021: Downstream Processing in Biotechnology Lecture Set 1: Introduction to Downstream Processing
Guhan Jayaraman
Dept. of Biotechnology, IIT- Madras
12-13 January 2015
Syllabus for the course Role of downstream processing in biotechnology, classes of bio-
products, physicochemical basis of separation; characteristics of biological molecules; regulatory requirements, process strategies (3 lecs)
Physical separation procs: Cell Disruption, Filtration, Sedimentation, Centrifugation (5 lecs)
Enrichment Procs – Precipitation, Membrane Separations (3 lecs) Staged Separation Procs – Distillation; Liq-liq extraction (8 lecs) Chromatographic Separation Processes – Ion-Exchange, Reversed-
phase, HIC, Affinity; Gel-filtration; Modes of Elution chromatography; Linear and Non-linear chromatographic separation (10 lecs)
Processing of Inclusion bodies and Protein Re-folding (2 lecs) Electrophoretic separation processes (3 lecs) Polishing Steps – Crystallization, Drying (5 lecs)
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Text and Reference Books Harrison R.G., Todd P., Rudge S.R., Petrides D.P., Bioseparations
Science and Engineering, Oxford Univ Press (2003) Belter P.A., Cussler E.L., Hu W-S, Bioseparation: Downstream
processing for Biotechnology, Wiley (1988). Ladisch M.R., Bioseparations Engineering: Principles, Practice and
Economics, Wiley Interscience (2001) Seader J.D. and Henley E.J., Separation Process Principles, Wiley
(2nd Ed., 2001) McCabe W.L., Smith J.C. and Harriott P., Unit Operations of
Chemical Engineering, Tata-McGraw Hill (7th Ed., 2005) Pauline Doran, Bioprocess Engineering Principles, Elsevier /
Academic Press (2nd Ed., 2013) Flickinger M.C. (ed.), Downstream Industrial Biotechnology:
Recovery and Purification, Wiley (2013)
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Timings, Exams, Grading… Classes on regular C slot timings on Mon, Tue, Wed Quizzes and ESE as per Institute schedule Quiz I, Quiz II : 15 marks each (total 30 marks) 3 Assignment-based Tests: 10 marks each (total 30 marks)
4th Feb , 4th Mar, 15th April End-Semester Exam: 40 marks Grading policy: Relative grading, subject to absolute lower cut-off for
‘E’ grade (30 marks) and ‘S’ grade (90 marks); Assignments will not be graded (contact TA for solutions); there will be 3 Assgn-based Tests
Institute policy will be enforced for Attendance and Unfair means: Please DO NOT give proxy-attendance; please be punctual TAs for course: D. Sreeja (Lab BT216); Contact her for timings
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General Types of Processes in Chemical and Biotechnology Process Industries Raw Material Processing
Mechanical Operations (Crushing, Grinding etc.)
Physico- Chemical Operations (Removal of Insolubles, Enrichment, Sterilization etc.)
Chemical and Bio-Conversions (Reactors; Fermenters)
Downstream Processing Separation Processes (Purification)
Finishing Operations (Product Formulation, Packaging etc.)
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Typical Unit Operations used in manufacture of Enzymes
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Generalized Downstream Processing Schemes
Role of Downstream Processing in Biotechnology Process Design and Economics
Design of separation processes to reduce process costs Reduce number of unit operations (optimal sequencing) Optimize each step (for product recovery, equipment size and
operating costs) Process should be easily scalable, reproducible and robust
Purification to meet regulatory requirements (esp. Biopharma Indus.) Achieve desired purity of product Maintain (biological) characteristics of the product Removal of (toxic) impurities to desired level Product and Process Validation (Analytics are critical !)
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Downstream Process Design depends on
Physico-chemical and biological characteristics of the product
Source from which product is obtained
natural source, fermentation product
End-use of the product
purity requirements, direct human consumption (food, therapeutics
etc.)
Regulatory issues
Market factors
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Bioproduct categories
Two main characteristics are: Size (MW) and the ability to withstand harsh environments during downstream processing
Small biomolecules Primary metabolites produced during growth (e.g. amino acids) Sugars
Sucrose: sugarcane, sugar beets (extraction, crystallization) Fructose: by glucose isomerase Glucose: amylase treatment of starch
Organic acids, alcohols, ketones Anaerobic fermentation
Vitamins Organic synthesis Plant sources and microbial fermentation
Secondary metabolites – produced by fungi, bacteria and plant tissues
during stationary phaseantibiotics such as penicillin; alkaloids from plant tissue culture
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Purity is not critical for non-human consumption
Biotech products market
Reference: Textbook (Harrison, et al., Bioseparations Science and Engineering)
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Costs of Downstream Processing in Biotechnology
Product Approx. Relative Price
% Downstream Processing Cost
Ethanol 1 15
Yeast Biomass 2 20
Citric Acid 3.5 30 - 40
Xanthan Gum 20 50
Penicillin G 60 20 - 30
Bulk Enzymes 100 40 - 65
Therapeutic Proteins
> 500 60 - 80
Downstream processing costs are mediated by product concentration in starting material, purity requirements, ease of separation, scale of operation and regulatory requirements
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Dose Purity Relationship
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Typical product profile during downstream processing of antibiotics
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Stage Typical Process Product Conc. (g/l)
Quality (%)
Harvest Broth Fermentation 0.1 – 5 0.1 – 1.0
Removal of insolubles Filtration 1 - 5 0.2 – 2.0
Isolation Extraction 5 - 50 1 - 10
Purification Chromatography 50 - 200 50 - 80
Polishing Crystallization 50 - 200 90 - 100
From Belter et al., Bioseparations, Chap. 1
Stages in a typical downstream processStage Objectives Unit Operations
Separation of insolubles
Remove cells / cell debris, other particulates
Filtration, centrifugation, sedmentation
Isolation of product / Primary recovery / Enrichment of product
Concentration of product; Removal major impurities and impurities having biggest property difference
Ultra-filtration, Precipitation, Extraction, Adsorption
Purification Removal of impurities with similar properties involving high-resolution techniques
Chromatography; Affinity separation methods; Electrophoresis
Polishing / Stabilization / Formulation
Removal of liquids; De-salting; Buffer exchange; Drying / Crystallization of product
Gel-filtration; Crystallization; Drying / Lyophilization
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Low resolution-high throughput
• Cell disruption
• Precipitation
• Centrifugation
• Ultra-filtration
• Extraction
High resolution - low throughput
• Ultracentrifugation
• Adsorption
• Chromatography
• Affinity Separations
• Electrophoresis
Unit Operations involved in Bioseparations
Downstream Process Design Heuristics Remove the most plentiful impurities first (e.g. cells, extracellular broth)
Remove the easiest-to-remove impurities first (e.g cells) and those which
may cause maximum damage to the product (e.g. proteases)
The most difficult and expensive separations should come at a later stage
Select processes that make use of the greatest differences in the
properties of the product and its impurities
Select and sequence processes that exploit different separation driving
forces
(Harrison et al., Bioseparations Science and Engineering, Chap 11)
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Principal Ingredients of Engineering Analysis Material Balances Chemical Thermodynamics: Phase Equilibria Transport Phenomena: Flux Relationships
Measures of Process Efficacy Product recovery (yield) Product purity (purification factor) Product concentration (enrichment factor) Product efficacy (biological activity, stability etc.) Product throughput Process cost per unit of product purified
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Quantifying Process Effectiveness
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In addition to the quantifiable measures of process efficacy…A separation process should also be
Easy to validate using online and offline analytics
Reproducible very similar profiles over several batches
Robust Should give reproducible results for minor variations at upstream
end of the process
Scalable From lab-scale to pilot and production-scale
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Product Analytics for Regulatory Compliance Identity Determination
HPLC, Immunoassay, Peptide mapping, amino-terminal sequencing Biological Activity and other Product characteristics
Bioassay (animal models, cell-culture); Biochemical assays (specific activity)
Glycosylation patterns; Immunogenicity Purity
Purity by RP-HPLC, electrophoresis; multimeric forms by IEX-HPLC; trace metals; host cell proteins; endotoxin; bioburden; sterility; particulates; moisture; volatile organics by GC; DNA by hybridization
Physical qualities Appearance, solubility, pH, content uniformity
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Analytical Tools Analysis of biological activity
Animal model assays Cell culture based assays In-vitro biochemical assays (e.g. enzymatic assay)
Analysis of Purity Electrophoretic Analysis: SDS-PAGE, Native Gel
Electrophoresis, Capillary electrophoresis, 2D gel-electrophoresis
HPLC, LC-MS
Impurities Protein Assays; Volatile Organics by GC; DNA hybridization Microbiological Assays (Sterility, Bioburden), Endotoxins (LAL
test), Viral assays (cell assays, ELISA, Nucleic acid probes), Phages (bacterial lysis test)
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For Process and Product Validation…
Detailed, Clear and Accurate Documentation
for every process step is Critical !
Finally… (and don’t ever forget this !)