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 bioproducts, 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, Reversedphase, 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)

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

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.)

Typical Unit Operations used in manufacture of Enzymes

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 !)

Downstream Process Design

depends
onbiological characteristics of the product
Physico-chemical and
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

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 phase
antibiotics such as penicillin; alkaloids from plant tissue culture

Purity is not critical for non-human consumption

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Biotech products market

Reference: Textbook (Harrison, et al., Bioseparations Science and Engineering)

Costs of Downstream Processing in

Biotechnology
Product
Approx. Relative % Downstream
Price

Processing Cost

Ethanol

15

Yeast Biomass

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
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

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Stages in a typical downstream

process
Stage
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; Desalting; Buffer exchange;

Drying / Crystallization of
product

Gel-filtration;
Crystallization; Drying /
Lyophilization

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Unit Operations involved in

Bioseparations
Low resolutionhigh throughput

High resolution
- low throughput

Cell disruption

Ultracentrifugation

Precipitation

Adsorption

Centrifugation

Chromatography

Ultra-filtration

Affinity Separations

Extraction

Electrophoresis
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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

Biological Activity and other Product characteristics

Bioassay (animal models, cell-culture); Biochemical assays

(specific activity)
Glycosylation patterns; Immunogenicity

Purity

HPLC, Immunoassay, Peptide mapping, amino-terminal

sequencing

Purity by RP-HPLC, electrophoresis; multimeric forms by IEXHPLC; 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

Analysis of Purity

Animal model assays

Cell culture based assays
In-vitro biochemical assays (e.g. enzymatic assay)
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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Finally (and dont ever forget this !)

For Process and Product Validation
Detailed, Clear and Accurate Documentation
for every process step is Critical !

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