Materials Related to Chp1

Todays meeting will cover: Bioseparation: what is it?

Overview of bioproducts: primary & secondary

metabolites, macromolecules, particulates

Introduction to Principles of Engineering Analysis in

Bioseparation

Materials related to Chp1

BTE4481 (Separation Processes II) Sem. 1, 2015-2016

where the cell mass from the upstream are processed to meet purity and quality requirements. Unit operation in downstream processing is

usually divided into several stages such as cell /harvest/disruption, product isolation, purification and polishing.

Introduction to Bioseparation:

Bioproduct of interest

upstream processes

e.g. fermentation Cell isolation and cultivation, cell banking and culture expansion of the cells until final harvest; also concerns with inoculum development, media development, improvement of inoculum by genetic engineering process, optimization of growth kinetics.

downstream processes

purification or bioseparation

BIOSEPARATION Processes:

Isolation, Separation, Purification & polishing of bioproducts

Plant tissues

Animal tissues

Immobilized

biocatalysts

Cells in bioreactor

bioproducts

BIOSEPARATION PROCESSES

extracted, purified and market ready bioproduct

Bioseparation challenge: to remove process & product contaminants & impurities from desired bioproduct i.e. to efficiently & economically recover a high purity bioproduct from a complex mixture of

related & functional molecules, impurities & contaminants which may have similar physical &

chemical properties

Slide credit to: Professor Ian Marison, Head of School of Biotechnology, Dublin City University

IMPURITIES components required for production and/or purification, but found in purification step

host cells, host cell proteins, nucleic acids, lipids

separation components (extractants; salts; urea; antifoam; etc..)

degraded products (misfolded; aggregated)

CONTAMINANTS components which may enter production process

foreign cells (bacteria, viruses) endotoxin (bacteria lipo-polysaccharides) extractables from membranes/filters/resins cleaning chemicals

Bioseparation: separation and purification of bioproducts A sequence of recovery and separation steps to: maximize the purity of the bioproduct minimize the processing time minimize yield losses minimize costs Continuous improvements required to remain competitive!

BIOPRODUCTS

Produced by Living Organisms

enormous range of physical & chemical properties enormous range of product values & production levels

3 CATEGORIES OF SOURCES:

Whole cells: single-cell protein, bakers yeast and animal feed supplements derived from yeast fermentation

Intracellular macromolecules: protein in cytoplasm, protein in

inclusion bodies from recombinant bacterial fermentations,

starch in inclusion bodies found in plant cells and intracellular

proteins

Extracellular products: proteins, antibiotics, organic acids, and

alcohols secreted during microbial fermentations or cell culture

Bioproduct Examples Molecular Weight

(Da)

Typical radius

Small molecules {can not be sedimented,

but can be separated by

extraction}

Sugars

Amino acids

Vitamins

Organic acids

200-600

60-200

300-600

30-300

0.5 nm

0.5 nm

1-2 nm

0.5 nm

Large molecules {highly adsorptive}

Proteins

Polysaccharides

Nucleic acids

103-106

104-107

103-1010

3-10 nm

4-20 nm

2-1,000 nm

Particles {can be collected by

sedimentation or filtration}

Ribosomes

Viruses

Bacteria

Organelles

Yeast cells

Animal Cells

25 nm

100 nm

1 m

1 m

4 m

10 m

Table 1.2 (Harrison et al, 2003)

Broad Categories of Bioproducts & their Sizes

PRIMARY Metabolites formed during the primary growth phase of the organism key metabolic intermediates in catabolism & anabolism

(A) Sugars: monosaccharides, disaccharides & polysaccharides

as products in bioprocess, examples:

- mannitol to fructose (partial oxidation by acetic acid bacteria)

- glucose to fructose (glucose isomerase)

where fructose corn syrup: sweeteners in soft drinks


(B) Organic alcohols, acids, and ketone

- can be produced by the anaerobic fermentation of microbes

- Examples: ethanol, isopropanol, acetone, acetic acid, lactic

acid, propionic acid


(C) Vitamins: e.g. Vitamin C

(ascorbic acid) Most vitamins can be synthesized in organic chemical reaction (may have harmful impurities) Extraction from plants and fermentation produces vitamins naturally (may incur at higher cost )

example of a present-day market tension: natural vs synthetic


(D) Amino acids

* Building blocks of proteins

* Specific properties of amino acid side group (review)

Acidic or basic AAs: adsorption by ion exchange or separation by electrophoresis

Many aliphatic side chains: preferential adsorption onto or extraction into nonpolar media

Free SH (sulfhydryl) Histidine


(E) Lipids

any fat-soluble (hydrophobic), naturally-occurring molecules, that are insoluble in water but soluble in nonpolar organic solvents. - highly extractable into nonpolar solvents

Triglycerides Phospholipids Steroids Waxes

Primary Metabolites

Commercial Significance

Citric acid

Glutamic acid

Lysine

Nucleotides

Phenylalanine

Polysaccharides

Vitamins

Lactic acid

Formic acid

Fructose

Acetic acid

Various uses in the food industry

Flavour enhancer

Feed supplement

Flavour enhancers

Precursor of aspartame, sweetener

Applications in the food industry

Enhanced oil recovery

Feed supplements

Biodegradable polymers

Fine chemicals

Food, fermentation

Food (vinegar) and fine chemicals

(Standbury et al, 2000 and Table 1.4)

Some primary products of microbial metabolism

and their commercial significant

SECONDARY Metabolites not formed during the primary growth phase of the organism formed near the beginning of the stationary phase

not associated with primary growth cycle

generally more complex with multiple functional groups

Example:

- penicillin, produced by Penicillium species

- used as antibiotic

- 3 x 107 kg/yr ; US$120/kg ; US$4 billion/yr

Produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin.

-ketoglutarate + AcCoA homocitrate L--aminoadipic acid L-lysine + -lactam L-lysine, inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production.

Penicillin core structure, where "R" is the variable group.

MACROMOLECULE bioproducts Proteins Nucleic Acids Polysccharides

PROTEIN bioproducts

includes enzymes, hormones, clotting factors, antibodies natural or by recombinant gene technology

Example: - erythropoietin (EPOGEN), 30000 g/mol - 2 kg/yr ; US$80 million/g ; US$2 billion/yr

Recombinant protein expression

to get more protein production & also improve protein purification by designing

fusion protein (inserting removable tags at C- or N-terminus of polypeptide)

His6x tag ; Glutathione S-transferase tag (GST); Maltose binding protein (MBP)

Sometimes recombinant proteins produced as improperly folded entities known

as inclusion bodies

Must be solubilized

Must allow for proper refolding

Classification of Proteins according to prosthetic group

PROSTHETIC GROUPS IN PROTEINS & HYBRID MOLECULES

Prosthetic group: non-amino acid portion (organic & inorganic compounds)

of a conjugated protein

e.g. glycosylation: oligosaccharides conjugated post-translationally to specific

AAs (Ser, Asn, Thr)

help stability, catalytic function, binding specificity, solubility, targeting, & transport

Classification of Proteins according to function

Nucleic acids: RNA & DNA

polynucleotides: repeating units of nucleotides phosphodiester linkage between 3OH & 5OH of ribose or deoxyribose

DNA (DeoxyriboNucleic Acid) RNA (RiboNucleic Acid)

* DNA - chemically more robust than proteins * 2o structures are highly stable, being based on base-pairing hydrogen bond

Oligo-or polynucleotides with commercial potentials

1. Antisense:

* bind to mRNA and inactivate it

* Antisense therapy: in vivo treatment of a genetic disease by blocking

translation of a protein with antisense DNA or RNA

2. Ribozyme:

* can react with RNA and catalyze its cleavage at sequence-specific site

* Ribozyme specific binding for cleavage of viral or bacterial genes

3. Aptamer:

Aptamers are nucleic acid species that have been evolutionary engineered

through in vitro selection or equivalently, SELEX (systematic evolution of

ligands by exponential enrichment) to bind with high affinity to various

molecular targets such as small molecules, proteins, nucleic acids, and even

cells, tissues and organisms.

Aptamers offer the utility for biotechnological and therapeutic applications as

they offer molecular recognition properties

In addition to their discriminate recognition, aptamers offer advantages over

antibodies as they can be engineered completely in a test tube

POLYSACCHARIDES

Highest Mwt of all bioproducts in use

* Not necessarily linear like peptides & nucleic acids

* Numerous OH : possible branching via typical glycosidic linkage

* Most familiar polysaccharides: starch, glycogen, cellulose

Starch:

* Major storage polysaccharide in higher plants.

* Mixture of amylose (-1,4, 2025%) & amylopectin (-1,4 & -1,6 per 24-30 residues, 7580%) * Provides 80% of dietary calories in humans worldwide

Glycogen

* Major storage polysaccharide in animals.

* Long straight glucose chains (-1,4) & branched (-1,6) * More branched than starch

Cellulose (-1,4): most abundant in nature & linear unbranched structural homopolysaccharides

* Starch and glycogen are stored fuels

* Bacterial polysaccharides: component of a number of vaccines

* Microbial polysaccharide: xanthan gum, dextran, alginate,

pullulan

* Cellulose, agarose, dextran are used in media for bioseparation

PARTICULATES Subcellular particles, bacterial inclusion bodies, whole cells, insoluble

macromolecular aggregates

Generally purified by centrifuge

Some very small particles ultracentrifugation

SCP (single-cell protein): microbial cells grown for food or feed applications

(algae, bacteria, yeast, filamentous fungi)

Subcellular components Bacterial inclusion bodies (100-1,000 nm)

Ribosomes (25 nm)

Liposomes or natural vesicles (100 nm)

Natural hormone granules (200 nm)

Early bioseparation steps include flocculation, sedimentation, filtration

CELLS yeasts, bacteria, mammalian (RBC), polio virus cells

Allahu akbar

where the cell mass from the upstream are processed to meet purity and quality requirements. Unit operation in downstream processing is

usually divided into several stages such as cell /harvest/disruption, product isolation, purification and polishing.

Recall:

Introduction to Bioseparation

Bioproduct of interest

upstream processes

e.g. fermentation Cell isolation and cultivation, cell banking and culture expansion of the cells until final harvest; also concerns with inoculum development, media development, improvement of inoculum by genetic engineering process, optimization of growth kinetics.

downstream processes

purification or bioseparation

Fermentation parameter factors affecting the DSP

properties of MOs (safety, classification, morphology, thermal stability, tendency to flocculate, size, cell wall rigidity, ....) influence the filterability, sedimentation & homogenisation performance

location of the product (intracellular, deposited in vacuoles or inclusion bodies or excreted into the broth - or biomass itself) will define the initial separation steps and purification strategy.

stability of the product defines the need & kind of pretreatment for inactivation or stabilisation.

product, by-products and impurities as well any additions to the broth (e.g. antifoam) may form an interfacial layer in extraction steps, give peaks in chromatography, block membranes in ultrafiltration and analytical equipment; also salts and trace elements often have to be removed prior to pharmaceutical use.

nutrient medium residues (pesticides, herbicides, etc.)

Introduction to Bioseparations: Engineering Analysis

Stages of Downstream Processing

SLECTED PHYSICAL BASIS FOR BIOSEPARATION

density: centrifugation, settling

size: membranes, filtration, size exclusion chromatography

charge: IEC, electrophoresis

solubility: precipitation, crystallization

affinity: affinity chromatography

liquid partitioning: extraction

solid partitioning: adsorption, chromatography

hydrophobicity: HIC

Other considerations:

thermal stability, diffusivities, reaction rate constants, separation

thermodynamics

BASIC PRINCIPLES OF ENGINEERING ANALYSIS

Governing equations:

material balance, equilibria, flux (or transport phenomena)

Material balance

Accumulation = inflow outflow + amt produced amt consumed

Equilibria extraction process: partition coeff. K=y/x y: conc. of a separand in the extract phase x: conc. of the same separand in the raffinate (usually heavy) phase

adsorption process

equilibrium constant for chemical reaction: Keq=[C]/([A][B]) for A + B C CS: conc. in the adsorbent phase

C: conc. in the liquid phase (C: chemical species & S:adsorption site)

[CS]=Keq[C] linear adsorption equilibrium (valid at low conc.)

BASIC PRINCIPLES OF ENGINEERING ANALYSIS Flux relationships (transport phenomena)

Flux = coefficient driving force flux: flowing per unit area per unit time

driving force: gradient down which units flow

coefficient: permeability or the inverse of a resistance (properties of medium)

Ohms law: Je= CE Je: current density

C: electrical conductivity

E: electrical potential gradient

Ficks first law for diffusive flux due to a concentration gradient dc/dx in one dimension

D: diffusion coefficient property of the medium In some cases, calculable from the Stokes-Einstein equation for spheres

JD= -D(dc/dx)

D = kT/6a (k: Boltzman constant, T: absolute temp., : viscosity, a: particle radius)

PROCESS & PRODUCT QUALITY

Purity = Amount of product Amount of product + amount of total impurities

Fold purification = the ratio of the purity at any stage in the process to

the purity at the start of the purification process

Another measure of purity is

where units of biological activity are assayed by means of a

biological test, such as moles of substrate converted per second

per liter or fraction of bacterial cells killed

Specific activity= Units of biological activity

mass

Yield = Amount of product produced Amount of product in feed

PURIFICATION TABLE example

PURIFICATION TABLE example

https://novoprotein.wordpress.com/2012/09/29/the-purification-table/

Specific activity

(U/mg)

PURIFICATION TABLE

https://novoprotein.wordpress.com/2012/09/29/the-purification-table/

Table 1 from previous slide:- Volume (ml) this refers to the measured total solution volume at the particular stage in the isolation.

Total protein (mg) the primary measurement is of protein concentration, i.e. mgml-1, which is obtained using a protein assay. Multiplying the protein concentration by the total volume gives the total protein (i.e. mg/ml x ml = mg).

Total activity (units) the activity, in units ml-1, is obtained from an activity assay. Multiplying the activity by the total volume gives the total activity (i.e. units/ml x ml = units).

Specific activity (units/mg) the specific activity is obtained by dividing the total activity by the total protein. Alternatively, the activity (units/ml) can be divided by the protein concentration (mg/ml), in which case the ml cancels out, leaving units/mg.

Purification (fold) Fold refers to the number of multiples of a starting value. In this case it refers to the increase in the specific activity, i.e. the purification is obtained by dividing the specific activity at any stage by the specific activity of the original homogenate. The purification per step can also be obtained by dividing the specific activity after that step by the specific activity of the material before that step.

Yield (%) the yield is based on the recovery of the activity after each step. The activity of the original homogenate is arbitrarily set at 100%. The yield (%) is calculated from the total activity (units) at each step divided by the total activity (units) in the homogenate, multiplied by 100. The yield can also be calculated on a per step basis by dividing the total activity after that step by the total activity before that step and multiplying by 100. The efficiency of a step is calculated as:- Purification (for that step) x % yield (for that step) /100

CRITERIA USED FOR DEVELOPING AND

EVALUATING A BIOSEPARATION PROCESS

product purity cost of production as related to yield scalability reproducibility and ease of implementation robustness with respect to process stream variables

The integration of unit operations for the efficient synthesis

of bioseparation processes will be discussed in

Bioseparation Design

CRITERIA USED FOR DEVELOPING AND

EVALUATING A BIOSEPARATION PROCESS

A therapeutic protein can be 99.99% pure but still unacceptable if any pyrogen* is present

Industrial enzyme: enzyme product with practically any impurities that do not inhibit the activity of the product or endanger the user are allowed

(*Pyrogen: any substance that produces a fever)

Recall,

BIOSEPARATIONS: a sequence of recovery and

separation steps maximize the purity of the bioproduct

minimizing the processing time, yield losses and costs

PURITY REQUIREMENTS

depends critically on application/use

therapeutic proteins: regulated (in USA by FDA) - DNA levels < 100 picograms (1 x 10-10 g) per dose - virus < 1 per million doses - bacteria need to be undetectable on agar plate

citric acid: regulated by FDA if food grade - Cl- < 50 mg/kg - oxalic acid < 100 mg/kg - calcium < 200 mg/kg - water < 0.5%

Industrial Enzymes/chemicals gluco-amylase, subtilisin, cellulase (30 80% Pure)

Diagnostic Enzymes and Food Grade Chemicals (90 95% Pure)

Therapeutic biopharmaceuticals (>97% Pure)

PURITY of BioProducts and DSP

Slide credit to: Professor Ian Marison, Head of

School of Biotechnology, Dublin City University

Bioproducts Production Level and Price (Inverse r/ship betw. selling price & prod. level) (continuous improvements required to remain competitive!)

Sale of bioproducts (e.g. biopharmaceuticals) must recover cost of development, production, marketing & clinical trials (for human therapeutics)

BIOPHARMACEUTICAL ROUTE TO MARKET

Bioproducts: Sales & Future Production

Documentation of Pharmaceutical Bioproducts

The official documentation of all compounds sold for medication must appear in the U.S. Pharmacopeia

For each substance the following data are given: structural formula empirical formula potency packaging and storage reference standards labelling chemical identification methods and assays for identity and purity

Validations of these properties- responsibility of the manufacturer

Bioproduct Production Regulation

cGMP: current good manufacturing practices What are these?

Set of guidelines used for all areas of producing, purifying, packaging, etc. any bioproducts (e.g. drug & food) to ensure safety and quality of the product. Everything is controlled and documented

Materials release and use (logs), equipment operation and cleaning (SOPs, logs), batch production records (PBRs) Personnel are held individually responsible

Mandated by the US Food and Drug Act of 1976

FDA Code of Federal Regulations, Title 21, Parts 210&211 (21 CFR Parts 210 and 211)

Slide adapted from Professor Ian Marison, Head of School of Biotechnology, Dublin City University

Integrated USP & DSP Design criteria

Concentration Productivity (volumetric, specific) Yield/ conversion Quality Purity Sequence Glycosylation Activity (in vitro, in vivo)

Design criteria: pharmaceutical product Order of importance Quality Concentration Productivity Yield/ Conversion High added value products

Design criteria: bulk product Order of importance Concentration Productivity Yield/ Conversion Quality Low added value products

Documents

Materials Related to Chp1