The document discusses downstream processing (DSP) in biotechnology, which is critical for extracting and purifying products from microbial fermentation, particularly in pharmaceuticals. It covers various DSP techniques, such as cell harvesting, cell disruption, concentration, chromatography, and the issues surrounding protein stability and purification. Additionally, it highlights methods for improving separation and recovery of proteins, particularly recombinant human granulocyte colony-stimulating factor (rh-GCSF) from E. coli.

1. DURGADEVI S
(16MG33)
M.TECH BIOTECHNOLOGY
PSG COLLEGE OF TECHNOLOGY
HARVESTING AND
DOWNSTREAM PRODUCT
PURIFICATION

2. Introduction
2
 Extraction and purification of product from
fermentation is known as Downstream
Processing ( DSP) or Product Recovery
 It is an essential step in the manufacture of
pharmaceuticals product
 Cost of the product is determined by the DSP
involved

3. [
3 Source - Glick
Upstrea
m
process
Downstream
process

4. 4
Source – Nandakisor et.a

5. 1. Harvesting Microbial Cells
5
 Separation of cells from the culture
medium solid-liquid separation
 Typical operations to achieve this:
 Filtration
 Centrifugation
 Sedimentation
 Flocculation
 Gravity settling

6. Membrane filtration
Dead end filtration Cross flow filtration
6 Source - Glick

7. Types of Filtration
7
 Based on the particle sizes to be separated
Source – Nandakisor et.al.

8. Centrifugation
8
 Principle - density differences between the
particles to be separated
 Separating solid particles from liquid phase
Source – Nandakisor et.al.

9. 2. Disrupting Microbial Cells
9
Source – Nandakisor et.al.

10. Mechanical cell disruption
10
Source - Glick

11. Disadvantages
11
 Whole cell contents are released out which
makes it difficult to separate out product of
interest from the mixture.
 Cell lysis increases viscosity of the solution
making it difficult to process in the further steps.
 Product released into harsh environment causing
the product to lose stability or activity.
 Enzymatic cell-disruption in large scale can be
expensive

12. 3. Concentration
12
 The commonly used techniques for concentrating
biological products are,
 Evaporation
 Liquid-liquid extraction
 Membrane filtration
 Precipitation
 Adsorption

13. 4.Purification by chromatography
13
 Stationary phase – porous solid matrix
 Mobile phase
Source – Nandakisor et.al.

14. Affinity chromatography
Hydrophobic interaction chromatography
14

15. 5.Formulation
15
 Maintenance of activity and stability of a
biotechnological products during storage and
distribution
 Stabilizing additives - prolong the shelf life of
protein. (stabilizers include sugars , salts,
polymers and polyhydric alcohols)
 Proteins may be formulated in the form of
solutions, suspensions or dry powders

16. 16
Source - Vasiliki Tsakaloudi
et.al.,

17. Problems in DSP of rDNA
product
17
S.N
o
Problems Solutions
1 Protein stability - depends
on the susceptibility of the
protein for proteolytic
decomposition
•lon-minus mutants from E.Coli k12 strains
•Synthesis of fusion protein
2
Recombinant proteins are
often found as insoluble
aggregates in the
cytoplasm. These
accumulations of solid
insoluble proteins are
called inclusion bodies
•Force protein secretion
•Changing the specific properties of the target
protein
•Distribution of the charge
•Fusion of heterologous gene with soluble
protein
•Fusion of heterologous gene with chaperon
3
Separation of Cells and
Cell Disruption – loss of
the target protein
•kil-gene of the plasmid ColE1 may yield
complete lyses of the cells. kil-gene, under the
control of the lac-promoter may lyse the cells

18. Contd.
18
S.N
o
Problems Solutions
4 Localization – recombinant
protein may be lethal to the
host when overproduced in
cytoplasmic region
•Target protein coupled with Signal sequences
(malE, ompA ,phoA )
•Human growth hormone which accumulated
in the periplasm of E. coli was able to be
exported into the medium upon induction of
bacteriocin release protein BRP
5 Cleavage of fusion protein –
some recombinant protein
with fusion protein are toxic
to the cells
•Fusion protein has to be constructed with a
specific cleavage side. (cleavage proteins –
Thrombin, blood coagulation factor Xa,
enterokinase)
•Oligonucleotide linker
•Intein
6 Plasmid instability •Strong and inducible promoter
7 Modification of proteins for
improvement of separation
•Altering certain physico-chemical properties
of the target protein by means of sitedirected

19. Fusion protein
19
 Fusion proteins - protects the cloned gene
product from attack by host cell proteases
Source - Glick

20. 20 Source - Glick

21. Cleavage of fusion protein
21
 Oligonucleotide linker encoding the amino acid
sequence Ile-Glu-Gly-Arg can be joined to the
cloned gene.
 Following synthesis and purification of the fusion
protein, a blood coagulation factor called Xa -
release the target protein
Source - Glick

22. 22
 Additional purification
steps in order to
separate both the
protein and the fusion
protein from the
protein of interest.
 This system has been
used to purify α-1-
antitrypsin and basic
human fibroblast
growth factor
Source - Glick

23. High recovery
23
 Histidine-tagged protein - passed over an
affinity column of nickel–nitrilotriacetic acid -
eluted – imidazole
 Greater than 90% recovery

24. 24
 Human interleukin-2 gene - the marker peptide
sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
 Dual function of reducing the degradation of the
expressed interleukin-2 gene product and then
enabling the product to be purified.
 Single step by immunoaffinity chromatography
 Bovine intestinal enterokinase

25. 25 Source - Glick

26. Secretion
26
 Directing a foreign
protein to the
periplasm or the
growth medium
makes its purification
easier and less costly
 Secretion into the,
 Periplasm
 Medium

27. Secretion into periplasum
27

28. Secretion into the medium
28
 Gram-negative bacteria can secrete a
bacteriocidal protein called a bacteriocin into the
medium.
 A bacteriocin release protein activates
phospholipase A
 Cleaves membrane phosopholipids so that both
the inner and outer membranes are
permeabilized.
 Some cytoplasmic and periplasmic proteins are
released into the culture medium

29. 29

30. 30

31. Objective
31
 In this paper they deal with different factors
influencing protein maturation and export, such
as
(i) the nature of the signal peptide (OmpA or PhoA),
(ii) the role of charge distribution near the leader
peptidase cleavage site,
(iii) the influence of chaperones (GroES and GroEL),
(iv) the incubation temperature and inducer
concentration
(v) the use of lysis proteins and
(vi) fusion to a known, secreted protein (preMBP)

32. 32

33. 33

34. Methods
34
 The plasmid-containing strains were grown in rich
medium at 37 ˚C Gene expression was induced by the
addition of 01-1 mM IPTG
 Cells in late exponential growth were harvested and
washed in TE
 The cell pellet was gently resuspended
 After 10 min at room temperature, the suspension was
centrifuged for 5 min at 6000g.
 The sucrose solution was carefully drained from the
tube and the pellet was resuspended in a same volume
of cold water
 The resuspended cells remained on ice for 10 min and
centrifuged at 15000 g for 10 min at 4 ˚C. The

35. 35
 The pellet was resuspended, freeze-thawed six
times and centrifuged for 5 min at 15000 g.
 The pellet and the supernatant are referred to as
the membrane and cytoplasmic fraction,
respectively. All samples were resuspended in
SDS loading buffer
 Analyzed by 14%SDS PAGE
 Visualized by immunodetection with monoclonal
antibodies directed against IL-2.

36. Result
36

37. Electron microscopy image
37

38. Reasons
38
 The signal peptides may have become buried
very soon after synthesis and were unable to
direct the protein to the export machinery
 Altered the charge distribution near the cleavage
site – Site-directed mutagenesis was performed
substitution lysines (K8/K9) of IL-2 by glutamic
acid (E), the K8E and substitution of cysteine
125 (C125) by alanine 125 (A125)
 Early folding or aggregation of the precursor
leads to loss translocation - chaperone factors,
GroEL, GroES, DnaK and SecB appear to be
required to prevent early protein folding

39. 39
Expression, secretion and purification of the
MBP-IL-2 hybrid protein

40. IL-2 bioassay
40
 Biological activity of human
IL-2 was tested using the
IL-2-dependent murine T
lymphocyte cell line CTLL-2
 Both recombinant proteins
were found to be active in
the assay. Interestingly,
 FXa cleavage yielded
aprotein with higher specific
activity which was very
 Similar to that of a
preparation of Chinese
Hamster ovary-derived
recombinant IL-2

41. 41

42. Introduction
42
 Human G-CSF - single chain polypeptide
containing 174 amino acid residues
(MW=18.8kDa, pI=6.1)
 One of the hemopoietic growth factors which
plays an important role in stimulating,
proliferation, differentiation, and functional
activation of blood cells.
 It contains a free cysteine at position 17 and two
intramolecular disulfide bonds

43. Objective
43
 To development of an efficient and scalable
procedure for production and purification of
recombinant human (rh-GCSF) of E. coli

44. Materials and methods
44
 Microorganism
E.Coli BL21 strain
 pET23 inducible
expression vector

45. Steps
45
 Batch cultivation
 Cell lysis and IB recovery – high pressure
homogenizer at 800bar
 Inclusion Bodies (IBs) washing –1Xtriton X -
centrifugation at 25–28°C for 30 min at 8000 g
 IB solubilization and refolding – urea
 Anion Exchange Chromatography - FPLC (Fast
Protein Liquid Chromatography)
 Detection - SDS-PAGE
 Conformation - Western blotting

46. Result and discussion
46

47. Western blotting
47

48. Conclusion
48
 2.2 g lˉ¹ rh-GCSF was produced in batch
cultivation with recovery yield about 40% and with
purity over than 99%.
 The process established in this study may be
functional in the recovery of other proteins
expressed in E. coli as cytoplasmic IBs.

49. References
49
1. Erwin Flaschel et.al., 1993 Improvement of Downstream
Processing of Recombinant Proteins By Means of
Genetic Engineering Methods Vol. 11, Pp. 31-78
2. http://www.biologydiscussion.com/biotechnology/downstr
eam-processing/stages-in-downstream-processing-5-
stages/10160
3. Bernard R. Glick, Jack J. Pasternak, and Cheryl L.
Patten. Molecular biotechnology : principles and
applications of recombinant DNA
4. Gabrielhea Lfmann et.al., 1993, Targeting of interleukin-2
to the periplasm of Escherichia coli, Journal of General
Microbiology, 139, 2465-2473.
5. S. Abolghasemi Dehaghani et.al., June 2010 An efficient
purification method for high recovery of Recombinant
Human Granulocyte Colony Stimulating Factor from
recombinant E.coli International Journal of
Environmental Science and Development, Vol. 1, 111-