This document provides an introduction to bioproducts and bioseparations. It defines key terms like bioseparations and bioproducts. Bioproducts can come from whole cells, intracellular macromolecules, or extracellular products from microbial fermentations or cell culture. The choice of separation method depends on the nature and properties of the product. Bioproducts are sold based on their chemical activity. The design of large-scale purification requires defining the product, starting material properties, and possible separation steps. Common unit operations are discussed for each of the four stages of bioseparations. Characterization and stability considerations for different types of bioproducts like proteins, nucleic acids, polysaccharides, and particulate products

1. PTT 302 DOWNSTREAM
PROCESSING TECHNOLOGY
SEMESTER 1 2013/2014
Lecture 1:
Introduction to Bioproducts and Bioseparation

2. 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

3.  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

4.  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

5. 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

6. 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.

8. 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

9. 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

10. 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)

11. Figure 1.1: Sucrose or table sugar is a disacharide composed
of the monosaccharides glucose and fructose

12. 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)

13. Figure 1.3: a-Amino acid structure showing
‘zwitterionic equilibrium at neutral pH.

14. Figure 1.4: Typical structures of three classes of compounds:
a fatty acid (stearic acid), a steroid (estradiol) and
prostaglandin (PGE2).

15. 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

16. 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)

17. 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.

18. 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’)

19. 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.

20. 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

21. 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

22. Figure 1.8: Three-dimensional
structure of ribonuclease
determined by x-ray diffraction
analysis

23. 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

24. Classification of proteins According to
Prosthetic Group

25. Figure 1.9: Structural
features of an antibody
(immunoglobulin G. or
lgG) Each pair of N-
terminal residues
constitutes an antigen
binding site.

26. Classification of Proteins According to
Function

27. Classification of Proteins According to
Function

28. Commercial Uses of Proteins

29. 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

30. 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

31. 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.

32. 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.

33. 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

34. Figure 1.12: The effect of pH on the relative activity of the enzymes
trypsin, cholinestrase, pepsin, and papain

35. 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

36. 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

37. Figure 1.13: The basic structures present in nucleic acids:
(a) pyrimidine bases, (b) purine bases,

38. (c) the nucleotide adenosine monophospate (AMP),

39. (d) a section of a DNA
chain

40. 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

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

42. Some Polysaccharides, Their Sources and
Uses

43. 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

44. Introduction to Bioseparations:
Engineering Analysis
a) Stages of Downstream Processing

45. Introduction to Bioseparations:
Basic Engineering Analysis
1. Material balance:
Accumulation = inflow –outflow + amount produced –
amount consumed
2. Equilibria:

46. Introduction to Bioseparations:
Basic Engineering Analysis
2. Equilibria:
Example: in extraction process that had gone to
equilibrium:

47. Introduction to Bioseparations:
Basic Engineering Analysis
2. Equilibria:
Example: in adsorption process that had gone to equilibrium:

48. Introduction to Bioseparations:
Basic Engineering Analysis
2. Flux (Transport Phenomena):
Example: Fick’s Law:

49. Introduction to Bioseparations:
Basic Engineering Analysis
2. Flux (Transport Phenomena):
Example: Darcy’s Law:

50. 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