Introduction to Downstream Processing

Downstream processing refers to the recovery and purification of biosynthetic

products, particularly pharmaceuticals, from natural sources such as animal or plant
tissue or fermentation broth

Removal of insolubles
involves the capture of the product as a solute in a particulate-free liquid

Product Isolation
is the removal of those components whose properties vary markedly from that of
the desired product.
Product Purification
is done to separate those contaminants that resemble the product very closely in
physical and chemical properties.
Product Polishing
describes the final processing steps which end with packaging of the product in
a form that is stable, easily transportable and convenient.
Down stream Processing of Proteins

Enzymology
 Proteins are the center of action in biological processes.
Nearly all the molecular transformations that define cellular
metabolisms are mediated by protein catalysis
One of the keys to deciphering the function of a given protein is
to understand its structure.
By examining how the amino acids are strung together, we can
endeavor to understand the chemical and physical properties of
proteins and, ultimately, their mechanism of action in living
organisms.
 Functions
• Diverse functions related to structure
– Structural components of cells
– Motor proteins
– Enzymes
– Antibodies
– Hormones
– Hemoglobin/myoglobin
– Transport proteins in blood
 Structure

• Amino acids
– Amino group
(NH2)
– Carboxyl group From: Elliott, WH. Elliott, DC. (1997) Biochemistry
and Molecular Biology. Oxford: Oxford University
(COOH) Press. p23
 Amino acids

• 20 amino acids make up

protein
• 8 essential amino acids
– 9 in infant (histidine)
From: Elliott, WH. Elliott, DC. (1997) Biochemistry and
• Dipolar
Molecular Biology. Oxford: Oxford University Press.
p23
– +ve end (NH3+)
– -ve end (COO-)
 Protein structure - bonding

• 5 bonds or forces determine structure

– Peptide bond
– Hydrogen bond
– Disulfide bond
– Ionic bond
– Hydrophobic force
 Peptide bond

• Peptide bond joins

amino acids
• Bond at both ends
– Increases range of
possible proteins

From: Elliott, WH. Elliott, DC. (1997) Biochemistry

and Molecular Biology. Oxford: Oxford University
Press. p23
Primary protein structure Primary structure of insulin

• Linear sequence of amino

acids forms primary
structure
• Sequence essential for
proper physiological
function

Bettelheim & March (1990) Introduction

to Organic & Biochemistry
(International Edition) Philadelphia:
Saunders College Publishing, p299
 Sickle cell anemia

• Replacement of
single glutamine
with valine in one
polypeptide chain
of hemoglobin
alters structure and
function Bettelheim & March (1990) Introduction to Organic & Biochemistry
(International Edition) Philadelphia: Saunders College Publishing, p301
 Secondary protein structure

• Peptide chains fold into secondary

structures:
–  - helix
–  - pleated sheet
– Random coil
  - helix

• Shape maintained
by hydrogen
bonds between
C=O and N-H
groups in
backbone
• R groups directed
outward from coil
From: Elliott, WH. Elliott, DC. (1997) Biochemistry and Molecular
Biology. Oxford: Oxford University Press. p28
 - pleated sheet

• Structure
maintained by
hydrogen bonds
between C=O and
N-H groups in
backbone
• R groups directed
above and below
backbone From: Elliott, WH. Elliott, DC. (1997) Biochemistry and Molecular
Biology. Oxford: Oxford University Press. p29
 Random coil
• Not really random
structure, just non-
repeating
– ‘Random’ coil has fixed
structure within a given
protein
– Commonly called
‘connecting loop region’
From: Elliott, WH. Elliott, DC. (1997) Biochemistry – Structure determined by
and Molecular Biology. Oxford: Oxford University
Press. p27 bonding of side chains
(i.e. not necessarily
hydrogen bonds)
Torsion angles of the polypeptide backbone

The Ramachandran plot

 Tertiary protein structure

• Secondary structures fold and pack together

to form tertiary structure
– Usually globular shape

• Tertiary structure stabilised by bonds

between R groups (i.e. sidechains)
Tertiary structure - H bond

• H bonds weak
allowing to be
broken and reformed
Hydrogen bond
easily
– Allows structural
change
• produces ‘functional’
molecules
Tertiary structure - disulfide bond

– Covalent bond
between sulfur
atoms on two
cysteine amino acids

From: Elliott, WH. Elliott, DC. (1997) Biochemistry

and Molecular Biology. Oxford: Oxford University
Press. p32
 Tertiary structure - ionic bond

• Ions on R
groups form salt
bridges through
ionic bonds

From: Summerlin, LR. (1981) Chemistry for the Life Sciences. New
York: Random House, p459
 Tertiary structure - hydrophobic forces

• Close attraction of non-polar

R groups through dispersion
forces
• Very weak but collective
interactions over large area
stabilise structure
• Repel polar and charged
molecules/particles Bettelheim & March (1990) Introduction to Organic & Biochemistry
(International Edition) Philadelphia: Saunders College Publishing, p302
 Quaternary protein structure

• Arrangement of multiple
tertiary structures into
single functional
complex
• Allows for changes in
structure/function in
response to chemical
stimuli From: Elliott, WH. Elliott, DC. (1997) Biochemistry and
Molecular Biology. Oxford: Oxford University Press. p27