Proses Hilir

(Downstream processing)
References
-Pharmaceitical Biotechnology (Gary Walls)
- Fermentation Technology (Stanbury )
- Sumber lain (on line)
 Pendahuluan
• Bioproses /Proses Fermentasi terdiri atas proses hulu
(Upstream) dan proses hilir (downstream)
• Proses hulu, pada umumnya meliputi :
- Optimasi galur / producer
- Optimasi media
- Optimasi kondisi proses (suhu, pH, aerasi
dan agitasi)
Proses Hilir
Proses hilir pada suatu fermentasi adalah proses untuk
mendapatkan produk akhir melalui tahapan isolasi,
pemekatan, purifikasi, dan formulasi.
Secara Umum, Proses hilir terdiri atas :
(a) Isolasi /recovery of product.
🡪dapat berupa produk intrasel (protein/enzim
dari sel);atau produk ekstrasel (metabolit (AA,
poliol) dari supernatan/ medium kultur, dsb.
(b) Purifikasi (fraksinasi /ultrafiltrasi, kromatografi)
(c) formulasi menjadi produk akhir.
Tahapan Proses Hilir Produk Intraseluler
• 1-Pemisahan biomassa (separation of biomass)
• 2-Pemecahan sel (cell disruption)
• 3-Pemekatan (concentration of broth)
• 4-Pemurnian awal (initial purification)
• 5-Pemurnian lanjutan (specific purification)
• 6-Pengeringan (de-watering)
• 7. Formulation
Tahap-tahap pada Proses Hilir
 Contoh lain : purifikasi protein
rekombinan (produk intraselular)
1. Recovery/Isolation produk awal)
• Proses isolasi awal produk intrasel berbeda dengan produk
ekstra seluler, yaitu:
Tahap ke-1) : Pengumpulan sel dgn sentrifugasi / microfiltrasi (sesuai
kebutuhan/skala)
- Sentrifugasi jika skala tidak terlalu besar( < 5L), pakai sel
bakteria.
Keuntungan: proses cepat (4000-6000 rpm,+ 15 menit)
Kelemahan : Investasi mahal (costly), ada kenaikan
temperature saat proses. Namun sudah ada yang
dilengkapi dgn control temperature/cool temp.
- Mikrofiltrasi: Jika skala cukup besar (> 10L), pakai sel kapang,
khamir atau bakteri berfilamen. Prosesnya lebih lama, tergantung
pada ukuran filter dan kekentalan cairan.
- Flokulasi : penggumpalan/pengendapan sel dgn garam anorganik
(jarang dilakukan)
Tahap ke-2 : Disrupsi/pemecahan cells dengan cara
mekanik(agitasi/homogenisasi), sonikasi, enzymatis
(Lysozime) atau bahan kimia (surfactan)
•Bahan kimia seperti detergen akan melisis dinding sel,
menginduksi protein denaturation , dan mempengaruhi tahap
purifikasi berikutnya. (hidrofobic interaction etc.)

• Metode mekanik spt :homogenisasi or agitasi kuat (dengan

Blender) bersama bahan yang abrasives (glassbeads) sering
digunakan untuk memecah sel setelah dipekatkan (5-10 kali).

•Metode pengeringan (drying): dgn cold aseton, lalu diekstraksi

2. Metode-metode pemecahan sel:
~3 macam teknik/cara: kimiawi, mekanik, enzimatik
 Extraction with Aqueous Two
Phase System (ATPS)

ATPS form when hydrophilic compounds such as some types of polymers

(polyethylene glycol, dextran, polypropylene glycol, etc.) and salts
(phosphates, sulfates, citrates, etc) are combined over certain critical
concentrations, resulting in the formation of two hydrophilic phases (Ionic
• Deep Eutectic Solvent (DES), adalah campuran dari dua atau
tiga komponen pada rasio molaritas tertentu sehingga
membentuk campuran eutektik dengan titik leleh yang lebih
rendah dari masing-masing komponen (Kalhor &
Ghandi,2019).
• DES memiliki karakteristik baik cairan ionik dan pelarut
organik, telah muncul sebagai generasi baru green solvent
dengan biaya rendah, efisiensi yang tinggi, dan karakteristik
yang dapat diterapkan secara tekno-ekonomis.
• Komponen DES menggunakan campuran hidrogen bond
acceptor (HBA) (seperti garam amonium kuaterner) dan
hydrogen bond donor (HBD) pada rasio molar yang sesuai dan
menghasilkan interaksi ikatan hydrogen dan interaksi Van
derWalls (E. L. Smith et al., 2014).
3. Penghilangan asam nukleat
Cara efektif : dengan rx. pengendapan atau dengan
penambahan enzim nuclease.
a. Dengan rx.pengendapan : Sejumlah molekul cationic
(bermuatan positiv) adalah precipitants efektiv bagi
DNA dan RNA; reaksinya terjadi melalui pembentukan
kompleks yg mengendap dgn muatan negativ asam
nucleat.
• Precipitant yang byk dipakai : poly-ethylenimine, a
long-chain cationic polymer.
• Selanjutnya endapan dipisahkan secara sentrifugasi or
filtrasi.
• Karena monomer dari polyethylenimine bersifat
karsinogenik maka proses pemisahannya harus
sempurna (completely removing any of the polymer
or its monomeric units that may remain in solution).
 .
b. Dengan penambahan enzim nuclease, yang
mengkatalis reaksi degradasi asam nukleat.
🡪nuclease treatment is quickly becoming the most
popular method of nucleic acid removal during
protein purification.
• Penggunaan nuklease ini efisien, dan relatif tidak
mahal. (e.g. nuclease from micrococcal)
• Berbeda dengan precipitant kimia, penggunaan
nuclease lebih aman (innocuous) dan tidak
bereaksi/bercampur dengan produk (final protein
product).
4. Pemekatan produk awal
• Pemekatan dilakukan dengan menginduksi pengendapan
produk menggunakan garam2, seperti amonium sulfat, atau
pelarut seperti etanol. Endapan diperoleh dgn Teknik
sentrifugasi/ filtrasi, lalu Endapannya dilarutkan kembali
dalam sejumlah kecil volume buffer yang sesuai (processing
buffer), atau
• Dengan Kromatografi penukar ion,IEC (in which proteins bind
to charged beads immobilized in a column), lalu dielusi
dengan perubahan konsentrasi garam.
• Kedua metode ini mampu menghasilkan tingkat kemurnian
yang tertentu/terbatas.
• Ultrafiltration, merupakan metode yang banyak digunakan
(the most common method) saat ini untuk pemekatan awal.
Ultrafiltrasi
• Sebelumnya, teknik mikrofiltrasi efektiv untuk menghilangkan
sel utuh dan pecahan sel (cell debris) dari larutan. Membran
filters yang dipakai berukuran diameter pori antara 0.1 - 10
μm. Ukuran pori tsb retaining whole cells and large
particulate matter, tapi meloloskan makromolekul spt
proteins. (Alele, N and Ulbricht, 2016)
• Membran ultrafiltrasi berukuran pori antara 1 - 20 nm,
sehingga mampu untuk menahan protein yang berukuran
kecil (low molecular mass).
• Membran ultrafiltrasi yang tersedia komersil memiliki
molecular mass cut-off antara 1 - 300 kDa.
• Membranes with molecular mass cut-off points of 3, 10, 30,
50, and 100 kDa are most commonly used.
 .• Fig : Lab scale
• Membran ultrafiltrasi tersedia Ultrafiltration separates
dari cellulose acetate or molecules based on size
cellulose nitrate. and shape
• Tersedia juga membran dari
polyvinyl chloride and
polycarbonate, yang lebih kuat
dan stabil secara kimia-fisika,
dan tidak menyerab protein.
(plastic-type membranes).
• Ultrafiltrasi skala lab seperti
pada gambar. The filter/flat
membrane is placed on a
supporting mesh at the bottom
of the cell chamber
• Bahan yang akan dipekatkan
dimasukan ke dalam “cell
system”, lalu dialiri gas inert spt
nitrogen.
 .
Ultrafiltrasi menjadi metode terpilih untuk pemekatan protein
karena :
•Metode ini sangat lunak, dengan efek samping yang kecil thd
bioaktivitas molekul protein.
•Perolehan kembalinya tinggi ( mencapai 99 %);
•Procesnya relatif lebih cepat (when compared with alternative
methods);
•Lebih efisien dan praktis (Little ancillary equipment is required).
Satu kelemahan dari technik filtrasi ini adalah kerentanannya thd
penyumbatan membran (rapid membrane clogging), terutama
yang cut off nya kecil. Sehingga diperlukan waktu yang lebih
lama (prolonged processing times).
 Chromatographic purification
• Setelah larutan protein dipekatkan , selanjutnya dilakukan
pemurnian hingga homogen (purified to homogeneity).
• Kromatografi Kolom mampu memisahkan protein satu dengan
yang lain berdasarkan perbedaan partisi masing-masing
protein thd fase diam dan fase geraknya. (a solid stationary
phase the chromatographic beads, usually packed into a
cylindrical column) and a mobile phase,usually a buffer).
• Idealnya, hanya target protein yang tertahan (retained) di
kolom, tapi sangat jarang/sulit terjadi.
• Setelah elusi sampel protein, lalu kolom dicuci dengan fase
gerak, untuk melepaskan/membebaskan protein yang terikat.
 .
• Komposisi fase gerak diubah
sehingga protein target
yang terikat dilepaskan
(desorption of the bound
protein).
• Fraksi-fraksi eluat
ditampung dalam tabung2
uji dan selanjutnya diperiksa
kadar dan aktivitas dari
total protein dan protein
target (lihat gambar).
• Fraksi protein target
ditampung dan dipakai
untuk proses purifikasi
selanjutnya.
•
.
Masing-masing protein punya karakteristik yang
membedakannya dari protein lainnya. Seperti : size and
shape, overall charge, the presence of surface hydrophobic
groups and the ability to bind various ligands.
• Berbagai metode kromatografi telah dikembangkan
berdasarkan karakteristik tsb sbb : (see Table ).
 .
• Penggunaan salah satu metode kromatografi ini biasanya
menghasilkan peningkatan aktivitas yang sangat tinggi (a
dramatic increase) pada protein target. A combination of
methods may be employed to yield highly purified protein
preparations (minimal 2 metode).
• Secara umum kombinasi 2- 4 metode dapat digunakan.
Gel-filtration and ion-exchange chromatography are amongst
the most common.
• Affinity chromatography is employed wherever possible
(needed), as its high biospecificity facilitates the achievement
of a very high degree of purification
 Size-exclusion chromatography
(gel filtration)
• Size-exclusion chromatography (gel filtration), disebut juga
gel-permeation or gel-filtration chromatography, separates
proteins on the basis of their size and shape.
• Saat sampel melalui kolom, protein yg berukuran besar tidak
dapat masuk ke celah/pori-pori gel sehingga cepat dielusi.
Protein yang berukuran kecil akan tertahan (beberapa lama)
dalam celah/pori-pori gel, sehingga terpisahkan.

• Umumnya, gel matrices utilized are prepared by chemically

cross-linking polymeric molecules such as dextran, agarose,
acrylamide and vinyl polymers. The degree of cross-linking
controls the average pore size of the gel prepared.
•
.
Sebagian besar gel yg disintesis dari polimer, tersedia dalm
berbagai ukuran pori.. The higher the degree of cross-linking
introduced, the smaller the average pore size.
• Gel dgn pori relative paling kecil dipakai untuk
menangkap/menahan molekul kecil (buffer dan garam2)
dalam pori matriks, dan meloloskan (exclude) semua protein,
sehingga dapat langsung ditampung sbg fraksi-fraksi protein.
(Figure 6.9).
• SEC jarang (rarely) digunakan di tahap awal
purifikasi protein.
• Small sample volumes must be applied to the
column in order to achieve effective resolution.
• Volume sampel biasanya 2–5 % dari volume
kolom. Furthermore, columns are easily fouled by
a variety of sample impurities.
• SEC lebih sering dipakai pada tahap akhir (the
end) purifikasi. (when the protein of interest is
already relatively pure and is present in a small,
concentrated volume).
 Ion-exchange
chromatography(IEC/KPI)
• Dasar pemisahan : perbedaan muatan molekul
protein-protein molecules; possess both positive and
negative charges, largely due to the presence of varying
amounts of amino acids. (N-terminal amino groups and the
C-terminal carboxy groups also contribute to overall protein
charge characteristics.)
• Several of the 20 amino acids that constitute the building
blocks of proteins exhibit charged side chains. At pH 7.0,
aspartic and glutamic acids have overall negatively charged
acidic side groups, whereas lysine, arginine and histidine
have positively charged basic side groups (see Figure ).
• The net charge exhibited by any protein depends on the
relative quantities of these amino acids present in the
protein, and on the pH of the protein solution.
 .
• The pH value at which a protein
molecule possesses zero overall
charge is termed its isoelectric point
(pI). At pH values above its pI, a
protein will exhibit a net negative
charge, whereas proteins will
exhibit a net positive charge at pH
values below the pI.
• Proteins may subsequently be
eluted by altering the pH or by
increasing the salt concentration of
the irrigating buffer. Ion-exchange
matrices that contain covalently
attached positive groups are termed
anion exchangers. These will adsorb
anionic proteins, e.g. proteins with
a net negative charge Ion-exchange chromatography is based upon
• Positively charged functional groups the principle of reversible electrostatic
(anion exchangers) include species such as attraction of a charged molecule to a solid
aminoethyl and diethylaminoethyl groups. matrix that contains covalently attached side
groups of opposite charge (Figure 6.11).
 .
• Matrices to which negatively charged groups are covalently
attached are termed cation exchangers, adsorbing cationic
proteins, e.g. positively charged proteins.
• Negatively charged groups attached to suitable matrices
forming cation exchangers include sulfo- and carboxy-methyl
groups (Table 6.3).
• During the cation-exchange process, positively charged proteins bind
to the negatively charged ion-exchange matrix by displacing the
counter ion (often H), which is initially bound to the resin by
electrostatic attraction.
• Elution may be achieved using a salt-containing irrigation buffer. The
salt cation, often Na+ of NaCl, in turn displaces the protein from the
ion-exchange matrix.
• The vast majority of purifi cation procedures employ at least one
ion-exchange step; it represents the single most popular
chromatographic technique in the context of protein purifi cation.
• Its popularity is based upon the high level of resolution achievable,
its straightforward scale-up (for industrial application), together with
its ease of use and column regeneration and relative least expensive.
• At physiological pH values most proteins exhibit a net negative
charge, Anion-exchange chromatography, therefore, is most
commonly used.
Hydrophobic interaction chromatography
• Of the 20 amino acids commonly found in proteins, eight are
classifi ed as hydrophobic, due to the non-polar nature of
their side chains (R groups, Figure 6.12).
 .
• A minority of hydrophobic amino acids, however, are present on the
protein surface and, hence, are exposed to the outer aqueous
environment.
• Different protein molecules differ in the number and types of
hydrophobic amino acid on their surface, and hence on their degree
of surface hydrophobicity. Hydrophobic amino acids tend to be
arranged in clusters or patches on the protein surface.
• Hydrophobic interaction chromatography fractionates proteins by
exploiting their differing degrees of surface hydrophobicity. It
depends on the occurrence of hydrophobic interactions between
the hydrophobic patches on the protein surface and hydrophobic
groups covalently attached to a suitable matrix.
• The most popular hydrophobic interaction chromatographic beads
(resins) are cross-linked agarose gels to which hydrophobic groups
have been covalently linked.
• Specific examples are octyl- and phenyl-sepharose gels, which
contain octyl and phenyl hydrophobic groups respectively
(see Figure)
• Protein separation by hydrophobic interaction chromatography is
dependent upon interactions between the protein itself, the gel
matrix and the surrounding aqueous solvent.
• Increasing the ionic strength of a solution by the addition of a
neutral salt (e.g. ammonium sulfate or sodium chloride) increases
the hydrophobicity of protein molecules.
• Protein samples, therefore, are best applied to hydrophobic
interaction columns under conditions of high ionic strength. As they
percolate through the column, proteins may be retained via
Hydro-phobic interactions. The more hydrophobic the protein, the
tighter the binding.
• After a washing step, bound protein may be eluted by utilizing
conditions that promote a decrease in hydrophobic interactions.
This may be achieved by irrigation with a buffer of decreased ionic
strength, inclusion of a suitable detergent, or lowering the polarity
of the buffer by including agents such as ethanol or ethylene glycol.
 Isolasi Metabolit Sekunder
• Metabolit sekunder pada umumnya adalah molekul kecil (low
MW, < 2kDa), dapat terkandung dalam biomassa atau medium
kultur, misalnya berupa antibiotic atau senyawa aktif lainnya.
• Secara praktis, tdp 3 metode isolasi yang dapat digunakan:
Ekstraksi dgn pelarut organic; Adsorpsi dengan penukar ion;
dan adsorpsi dengan resin penjerap.
• Sebelum isolasi, pastikan bahwa konsentrasi metabolit dalam
medium cukup tinggi (misal, yield = 40 g/L), jika perlu
dilakukan proses pemekatan terlebih dahulu.
• Tahap-tahap prosedur umum isolasi meliputi: identifikasi,
isolasi dan pemekatan(konsentrasi).
• Tahap Identifikasi: pemastian keberadaan metabolit
extrasel(di medium) atau intrasel (dalam sel); stabilitasnya thd
panas, dan hidrolisis.
• Tahap isolasi metabolit dari material biologi : (i)Isolasi dengan
pelarut organic yg sesuai (misal, methanol/etil asetat, dsb);
(ii)Optimasi kondisi ekstraksi, seperti pH, pelarut, resin
penukar ion yg cocok(asam/basa), (iii)Teknik pemekatan awal
yg cocok(liofilisasi/ adsorpsi)
• Pemekatan ekstrak dan eluat dari penukar ion :
🡪Dapat dilakukan dengan evaporasi pada temperature ckp
rendah( <60oC, dengan vacuum-evaporasi); atau liofilisasi
(freeze driying) dari larutan ber-air.
1. Ekstraksi : metabolit yg 2. Adsorpsi pd penukar ion
sdkt polar tdk larut air
dapat diekstraksi dgn
pelarut organic yang
sesuai. (Prinsip pemilihan:
Non-polar dissolved
non-polar)
pilihan PO : polar – non-polar
Recoveri pelarut : Destilasi
Affinity chromatography
• Affinity chromatography is often described as the most
powerful highly selective method of protein purifi cation
available. This technique relies on the ability of most proteins
to bind specifically and reversibly to other compounds, often
termed ligands (Figure 6.14).
 .
• A wide variety of ligands may be covalently attached to an inert
support matrix, and subsequently packed into a chromatographic
column. In such a system, only the protein molecules that
selectively bind to the immobilized ligand will be retained on the
column.
• Washing the column with a suitable buffer will flush out all
unbound molecules. An appropriate change in buffer composition,
such as inclusion of a competing ligand, will result in desorption of
the retained proteins.
• Elution of bound protein from an affi nity column is achieved by
altering the composition of the
• elution buffer, such that the affi nity of the protein for the
immobilized ligand is greatly reduced.
• A variety of non-covalent interactions contribute to protein–ligand
interaction. In many cases, changes in buffer pH, ionic strength,
inclusion of a detergent or agents such as ethylene glycol (which
reduce solution polarity) may suffice to elute the protein.