PTT 302 DOWNSTREAM

PROCESSING TECHNOLOGY

SEMESTER 1 2013/2014

Lecture 1:

Introduction to Bioproducts and Bioseparation

Definitions

Bioseparations

A sequence of recovery and separation steps – maximize the purity of the bioproduct while minimizing the processing time, yield losses and costs

Bioproducts

Chemical substances or combinations of chemical substances that are made by living things

Can be broadly classified into three categories of sources: Whole cells: single-cell protein, baker’s yeast and animal feed supplements

derived from yeast fermentation

Intracellular macromolecules: 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

The choice of separation method depends on the nature of the product – purity, yield and activity are the goals

Bioproducts are sold for their chemical activity, example:

Methanol for solvent activity

Ethanol for its neurological activity or as fuel

Penicillin for its antibacterial activity

Streptokinase (an enzyme) for its blood clot dissolving activity

The design of a large-scale purification process

requires three main considerations :

Clearly defining the final product objective (therapeutic for

human or animals, industrial enzymes)

Characterizing the starting material (from bacteria, yeast,

mammalian cell, knows the physicochemical properties of

the product such as surface hydrophobicity, pI, stability etc.)

Defining possible separation steps and constraints

regarding the protein product, operations and conditions to

be used

Rules of Thumb

In selection of separation sequence, five main heuristics or rules of thumb provide a good basis for process selection which are as follows:

Rule 1: Choose separation processes based on different physical, chemical or biochemical properties

Rule 2: Separate the most plentiful impurities first

Rule 3: Choose those processes that will exploit the differences in the physico-chemical properties of the product and impurities in the most efficient manner

Rule 4: Use a high-resolution step as soon as possible

Rule 5: Do the most harduous step last

Objectives and Typical Unit Operations of

the Four Stages in Bioseparations

Individual recovery operations can be grouped into different categories, depending on their general purpose.

The sequence of recovery operations that typically employed in practice is as follows:

Separation of insolubles. Insoluble materials include whole cells, cell debris, pellets of aggregated protein, and undissolved nutrients. Common operations for this purpose are sedimentation, centrifugation, and filtration.

Isolation and Concentration. Generally refers to the isolation of the desired product from unrelated impurities. Significant concentration is achieved in the early stages, but concentration accompanies purification as well. This category includes extraction, ultrafiltration, precipitation, and ion exchange.

Primary Purification. More selective than isolation; some purification steps can distinguish between species having very similar chemical and physical properties. Primary purification techniques include chromatography, affinity methods, and fractional precipitation.

Final Purification (Polishing). Necessitated by the extremely high purity required of many bioproducts, particularly pharmaceuticals and therapeutics. After primary purification the product is nearly pure but may not be in the proper form. Partially pure solids may still contain discolored material or solvent. Crystallization and/or drying are typically employed to achieve final purity.

Categories of Bioproducts and Their

Sizes

Bioproduct Examples Molecular Weight

(Da)

Typical radius

Small molecules Sugars

Amino acids

Vitamins

Organic acids

200-600

60-200

300-600

30-300

0.5nm

0.5nm

1-2nm

0.5nm

Large molecules Proteins

Polysaccharides

Nucleic acids

103-106

104-107

103-1010

3-10nm

4-20nm

2-1,000nm

Particles Ribosomes

Viruses

Bacteria

Organelles

Yeast cells

Animal Cells

25nm

100nm

1µm

1µm

4µm

10µm

Characterization of Biomolecules

Because biomolecules differ greatly in nature, different separation principles are required for their recovery and purification.

Their relative molecular masses vary generally, biomolecules are rather unstable and their stability depends on many different factors such as:

pH

Temperature

Ionic strength

Type of solvent used

Presence of surfactant

Metal ions

Small Biomolecules

can be divided into two categories primary metabolites and secondary metabolites Primary Metabolites formed during the primary growth phase of the

organism Sugar – monosaccharides, disaccharides or

polysaccarides (Figure 1.1) Organic alcohols, acids, and ketones – can be

produced by anaerobic fermentation of microorganism ex. Ethanol, isopropanol, acetone, acetic acid (Figure 1.2)

Vitamins – vitamin A (carotenoid), B, C (ascorbic acid), D (hormone D, steroid)

Figure 1.1: Sucrose or table sugar is a disacharide composed

of the monosaccharides glucose and fructose

Small Biomolecules (cont’d)

Amino acids – building blocks of proteins

specific properties of amino acid side groups can be

exploited in purification methods. A protein rich in

acidic @ basic amino acids on its surface can be

adsorbed by ion exchange or separated by

electrophoresis (Figure 1.3)

Lipids – natural fats consist of fatty acids, lipids,

steroids this family of bioproducts- highly extractable

into nonpolar solvents (Figure 1.4)

Figure 1.3: a-Amino acid structure showing

‘zwitterionic equilibrium at neutral pH.

Figure 1.4: Typical structures of three classes of compounds:

a fatty acid (stearic acid), a steroid (estradiol) and

prostaglandin (PGE2).

Some primary products of microbial metabolism and their

commercial significant (Standbury et al, 2000)

Primary Metabolites Commercial Significance

Ethanol

Citric acid

Glutamic acid

Lysine

Nucleotides

Phenylalanine

Polysaccharides

Vitamins

‘Active ingredient’ in alcoholic beverages

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

Secondary Metabolites

Not produced during the primary growth phase of a

microorganisms but at or near the beginning of the

stationary phase

Antibiotics – best known most extensively studied

Antibiotics: erythromycin, tetracycline and streptomycin

(Figure 1.5)

Figure 1.5:Structures of three well-known antibiotics: erythromycin, tetracycline,

and streptomycin. The choice of purification method is influenced by the profound

differences in the chemical structure of these compounds.

Macromolecules: Protein

Primary structure (the covalent amino acid sequence) (Figure 1.6)

established by the sequence of nucleotide codons of the messenger RNA for that protein

amino acid- held together in strictly linear sequence and all backbone bonds are covalent

no branched peptide, despite the existence of amino and carboxyl side chains capable of forming amine linkages

Secondary structure (the hydrogen-bonded structures) (Figure 1.7)

two main forms – the formation of hydrogen bonds between oxygen and nitrogen atoms in the peptide backbone

two structures that form are the α helix and the βsheet (or‘pleated sheet’)

Amino acid sequence of bovine insulin.The two polypeptide chains are held

together by disulfide bridges, but they originated from single chain, a

portion of which was excised by the cell.

A peptide chain in an α-helix

configuration, showing hydrogen

bonding (dashed lines) that stabilizes

the structure

Representation of three

parallel peptide chains

in a β-sheet structure.

Figure 1.7: Secondary Structure

Protein (cont’d)

Tertiary structure (the folding pattern of hydrogen-bonded and disulfide-bonded structures) (Figure 1.8) the three dimensional folding of coiled or pleated polypeptide chain establishes the following: surface properties, catalytic activity, stability, mechanical strength and shape

Quaternary structure (the formation of multimeric complexes by individual protein molecules)

the folded peptide chains interact with one another in the native conformation of an oligomeric protein ex. A complete hemoglobin molecule contains two α-globin and two β-globin peptide chains

maintained by intermolecular bonds, including ionic and covalent linkages

Figure 1.8: Three-dimensional

structure of ribonuclease

determined by x-ray diffraction

analysis

Protein (cont’d)

prosthethic group and hybrid molecules

conjugated proteins- contain not only amino acids but other organic and inorganic compounds

the non-amino acid portion of a conjugated protein- called prosthetic group

conjugated proteins- classified on the basis of the chemical nature of their prosthetic group

proteins containing lipids or sugar moieties – called hybrid compounds

antibodies – hybrid molecules – become a major product of biotechnology companies (Figure 1.9)

antibodies – globular proteins synthesized by B lymphocytes in animals

Classification of proteins According to

Prosthetic Group

Figure 1.9: Structural

features of an antibody

(immunoglobulin G. or

lgG) Each pair of N-

terminal residues

constitutes an antigen

binding site.

Classification of Proteins According to

Function

Classification of Proteins According to

Function

Commercial Uses of Proteins

Commercial Uses of Proteins

Application Commercial Enzymes

Animal Feed: Enzyme supplementation Bio-Feed Pro, Bio-Feed Wheat, Bio-

Feed Beta

Baking: Dough Improvement Fungamyl Super, Pentopan,

Lipopan™

Dairy: Milk protein modification Alcalase®, Pancreatic Trypsin Novo

(PTN), Flavourzyme

Detergent: Removal of fatty stains Lipolase®, Lipolase Ultra,

LipoPrime™

Starch: Starch liquefaction Termamyl, BAN, Fungamyl

Textiles: Wool finishing Esperase, Savinase

Stability of Protein

Stability of protein – temperature, pH, shear Protein degrade via several pathways: deamination of

asparagine and glutamine, oxidation of methionine, oligomerization, aggregation, denaturation (breakdown of hydrogen bonds and ionic bonds)

Temperature as it is increased, denaturation of a protein will start to occur at

some point Thermal denaturation for many proteins begins to occur at 45 to

500C Refer to Fig. 1.10 & 1.11; when the temp is high enough, the hydrogen

bonds are broken (dotted line in fig. 1.10 and the b-pleated sheet structure of the protein is disrupted)

Some proteins – still active at temp much higher than 45 to 500C ; ex. Enzymes isolated from thermophilic bacteria inhabiting hot springs – active at temp above 850C

Figure 1.10: Schematic drawing showing the conformational change that occurs when

ribonuclease is heated above its thermal denaturation temperature. Dotted lines indicate

hydrogen bonds, and numbers indicate the positions of the amino acid cysteine. which forms

disulfide bonds between chains.

Figure 1.11: Thermal denaturation of ribonuclease as measured by ∆A 287 , the change in absorbance at 287 nm compared with a reference solution at zero absorbance.

Protein (cont’d)

pH

often varied during the d/stream processing of proteins

Fig 1.12 illustrates the phenomenon for four enzymes for which effect of pH on the relative activity of the enzymes has been determined

In these cases the pH changes- leading to a change in the ionization state and structure of the active site

At extreme of pH, proteins are not only denatured but can be completely hydrolysed to their constituent amino acids

At extreme of pH, proteins are not only denatured but can be completely hydrolysed to their constituent amino acids

Figure 1.12: The effect of pH on the relative activity of the enzymes

trypsin, cholinestrase, pepsin, and papain

Protein (cont’d)

Shear

Not significant except when gas-liquid interfaces are present

Ex. In a rotating disk reactor that had a gas phase caused by incomplete

filling of the reactor

This loss of activity – attributed to the proteins being adsorbed to the

gas-liquid interfaces in an unfolded partially active and inactive state

If the turbulence in the solution – sufficiently high, the interface with

protein adsorbed breaks, causing unfolded protein to be inactivated

Therefore, it is important – gas-liquid interfaces be avoided/ minimized

Macromolecules: Nucleic acids and

Oligonucleotides

Nucleic acids- polynucleotides whose primary structure consists of repeating units of nucleotides (Figure 1.13)

A nucleotide consists of a nitrogeneous base, a ribose or deoxyribose sugar and one or more phosphate groups

Ribonucleotide acid (RNA) – polynucleotide containing only ribose sugar while deoxyribose (DNA) contains only deoxyriboses (DNA) (lacking a hydroxyl group at the 2’ position)

The use of DNA in tiny quantities; large-scale purification – never been necessary

However, a new family of nucleic acid products – currently arising

Commercial potential of oligo- and polynucleotides are antisense sequences, ribozymes and aptamers (combinatorial products)

These three categories of nucleic acid products- targeted for the therapeutics and diagnostics field

Figure 1.13: The basic structures present in nucleic acids:

(a) pyrimidine bases, (b) purine bases,

(c) the nucleotide adenosine monophospate (AMP),

(d) a section of a DNA

chain

Macromolecules: Polysaccharides

Of all bioproducts in use; the highest MW and the longest end-to-end polymer length (Figure 1.14)

Most familiar- starch, glycogen and cellulose

Most of them- extracted from plants, several are produced microbially; ex. Dextran, alginate

Highly purified- seldom sold, sources and uses of selected p/saccharides are listed in Table 1.6

Figure 1.14: Two polysaccharides. (a) Amylose, (b) Chitin

Some Polysaccharides, Their Sources and

Uses

Particulate Products

The form of sub-cellular particles, bacterial inclusion

bodies, whole cells and insoluble macromolecular

aggregates

Purifying of suspended animal and plant cells- to

obtain pure populations for biochem. study and for

food/ feed applications, starting material for pure

bioproducts

Introduction to Bioseparations:

Engineering Analysis

a) Stages of Downstream Processing

Introduction to Bioseparations:

Basic Engineering Analysis

1. Material balance:

Accumulation = inflow –outflow + amount produced –

amount consumed

2. Equilibria:

Introduction to Bioseparations:

Basic Engineering Analysis

2. Equilibria:

Example: in extraction process that had gone to

equilibrium:

Introduction to Bioseparations:

Basic Engineering Analysis

2. Equilibria:

Example: in adsorption process that had gone to equilibrium:

Introduction to Bioseparations:

Basic Engineering Analysis

2. Flux (Transport Phenomena):

Example: Fick’s Law:

Introduction to Bioseparations:

Basic Engineering Analysis

2. Flux (Transport Phenomena):

Example: Darcy’s Law:

Introduction to Bioseparations:

Engineering Analysis (cont’d)

Criteria should be used in evaluating and developing 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