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 !)