3 Downstream Process Farmasi

8/12/2019 3 Downstream Process Farmasi

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Downstream Processing in

Biopharmaceutical Fermentation

Alwani Hamad, ST, MSc.

Fakultas Farmasi

Universitas Muhammadiyah Purwokerto

Teknologi Fermentasi

MK: Bioteknologi


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2

Course content

Persyaratan proses fermentasi

Kondisi dan variable fermentasi

Fermenter/ bioreaktor

Media dan optimasi media untuk fermentasi Kinetika pertumbuhan bakteri untuk menghitung hasil

produk fermentasi

Downstream process hasil fermentasi (produk

biopharmacy)Evaluasi : Ujian (closed book)

Buku rujukan:G Rao.2007. Introduction to Biochemical Engineering. Tata Mc Graw- Hill Publishing

Company Limited

Stanbury, Whitaker and Hall, 2003. Principles of Fermentation Technology Butterworth


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Typical Production Process Flow

(Feed 2)

(Feed 3)

(Feed 4)

Chrom 1Chrom 3

Cryo-preservation

Concentration /

Diafiltration

Centrifuge

Viral Removal

Filtration

(Feed1)Inoculum Expansion

(Spinner Bottles)Ampule Thaw

Chrom 2


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Media Prep

Working Cell

Bank

Sub-

Culture

Inoculum

Sub-

Culture

Sub-

Culture

Sub-

Culture

Sub-

Culture

Large Scale Bioreactor

Wave

Bag

Seed Bioreactors

Fermentation

150L

Bioreactor

750L

Bioreactor

5,000L

Bioreactor

26,000L

Bioreactor

Depth

Filtration

Collection

Centrifuge

Harvest/Recovery

Harvest

Collection

Tank

1,500L

FilterChromatography

Skid

Anion Exchange

Chromatography (QXL)

Column EluateHold

Tank

8,000L

Eluate

Hold

Tank

6,000L

Filter

ChromatographySkid

Protein A

Chromatography

Column

Chromatography

Skid

Column

Eluate

Hold

Tank

20,000L

Hydrophobic Interaction

Chromatography (HIC)

Eluate

HoldTank

20,000L

Viral

Inactivation

Eluate

Hold

Tank

5,000L

Filter

ChromatographySkid

Anion Exchange

Chromatography(QFF - Fast Flow)

Column

Post-viral

Hold

Vessel

3,000L

Viral Filtering Ultra Filtration

DiafiltrationBulk

Fill

Purification

24 days 31 days

8 days

1 day

Upstream/Downstream Manufacturing Overview


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Hasil proses fermentasi

Mikroorganisme

Product

Whole cells Cell debris/fragments

Soluble and insoluble medium product

Proteins Undissolved nutrient components

biomass


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Downstream process dalam

Fermentasi Langkah proses downstream merupakan salah satu langkah dalam proses

fermentasi yang sangat penting yaitu langkah setelah proses fermentasi

untuk menghasilkan bioproduk dengan menggunakan unit operation agar

diperoleh produk yang terpisah dari campuran yang lain

Dalam downstream process : cost 2060% dari total cost proses. >90%

untuk produk rekombinant DNA

Downstream processing steps = operasi pemurnian produk hasil

fermentasi

Langkahlangkah dalam downstream process

Initial isolation

Product recovery

Purification and concentration


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Jenis unit operation yang digunakan

dalam downstream process Lihat table 17.1 hal 158 Rao Introduction of biochemical engineering

Dapat dibagi sebagai berikut :

Pemisahan suspended solid

Filtrasi

Sedimentasi

Centrifugation

Foam separation

Precipitation

Cell Disruption

Pemisahan satu fasa Ekstraksi

kromatografi


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Cell disruption

Mechanical method

High speed agitation

Grinding with abrasives

High pressure pumping (homogenization)

Non-mechanical method

Osmotic shock

Treatment with solvent and detergents

Freezing and thawingf

Enzymatic digestion of cell walls


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Know the Characteristics of

Your Protein

Green Fluorescent Protein

(GFP)

Sequence of Amino AcidsMSKGEELFTGVVPVLVELDGDVNGQKF

SVSGEGEGDATYGKLTLNFICTTGKL

PVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDD

GNYKTRAEVKFEGDTLVNRIELKGID

FKEDGNILGHKMEYNYNSHNVYIMG

DKPKNGIKVNFKIRHNIKDGSVQLAD

HYQQNTPIGDGPVLLPDNHYLSTQS

ALSKDPNEKRDHMILLEFVTAARITH

GMDELYK

Tertiary Structure

Contoh downstream process dalam

industri biopharmacy


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Know the Characteristics of Your Protein

Green Fluorescent Protein (GFP)

MW (molecular weight = 27,000 Daltons (27 kD)

pI (isoelectric point) = 4.8 Hydropathicity (=hydrophobicity) =


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Tissue Plasminogen Activator

MW 60 kD pI: 8.04 Hydrophobicity -.516

Human Serum Albuminmkwvtfisll llfssaysrg vfrrdthkse iahrfkdlge ehfkglvlia fsqylqqcpf

61 dehvklvnel tefaktcvad eshagceksl htlfgdelck vaslretygd madccekqep

121 ernecflshk ddspdlpklk pdpntlcdef kadekkfwgk ylyeiarrhp yfyapellyy181 ankyngvfqe ccqaedkgac llpkietmre kvltssarqr lrcasiqkfg eralkawsva

241 rlsqkfpkae fvevtklvtd ltkvhkecch gdllecaddr adlakyicdn qdtissklke

301 ccdkplleks hciaevekda ipenlpplta dfaedkdvck nyqeakdafl gsflyeysrr

361 hpeyavsvll rlakeyeatl eeccakddph acystvfdkl khlvdepqnl ikqncdqfek

421 lgeygfqnal ivrytrkvpq vstptlvevs rslgkvgtrc ctkpesermp ctedylslil

481 nrlcvlhekt pvsekvtkcc teslvnrrpc fsaltpdety vpkafdeklf tfhadictlp

541 dtekqikkqt alvellkhkp kateeqlktv menfvafvdk ccaaddkeac favegpklvw

601 stqtala

MW 69 kD pI 5.82 Hydrophobicity -.395

1 rrgarsyqvi crdektqmiy qqhqswlrpv lrsnrveycw cnsgraqchs vpvkscsepr 61cfnggtcqqa lyfsdfvcqc pegfagkcce idtratcyed qgisyrgtws taesgaectn 121

wnssalaqkp ysgrrpdair lglgnhnycr npdrdskpwc yvfkagkyss efcstpacse 181

gnsdcyfgng sayrgthslt esgasclpwn smiligkvyt aqnpsaqalg lgkhnycrnp 241

dgdakpwchv lknrrltwey cdvpscstcg lrqysqpqfr ikgglfadia shpwqaaifa 301

khrrspgerf lcggilissc wilsaahcfq erfpphhltv ilgrtyrvvp geeeqkfeve 361 kyivhkefdd

dtydndiall qlksdssrca qessvvrtvc lppadlqlpd wtecelsgyg 421 khealspfys

erlkeahvrl ypssrctsqh llnrtvtdnm lcagdtrsgg pqanlhdacq 481 gdsggplvcl

ndgrmtlvgi iswglgcgqk dvpgvytkvt nyldwirdnm rp

Some Other Proteins of Interest


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Clarification or

Removal of Cells and Cell Debris

Using Centrifugation

Using Depth Filtration


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Control Panel

Cut-away view

Protective enclosure

Basic components of a centrifuge

Door

Rotor

Drive

shaft

Motor

Centrifugal force

Sedimentatio

n

path of

particlesPellet

deposited

at an angle

Centerofrotation

rminimum

raveragermaximum

Centrifuge

An instrument that generates centrifugal force.

Commonly used to separate particles in a liquid from the liquid.


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Continuous Centrifugation

Media and Cells In & Clarified Media Out


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Separation of particles from liquid by applying

a pressure to the solution to force the solution through a

filter. Filters are materials with pores.

Particles larger than the pore size of the

filter are retained by the filter.

Particles smaller than the pore size of the filter pass

through the filter along with the liquid.

Filtration


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Traps contaminants larger than the pore size on the top surface of the membrane.Contaminants smaller than the specified pore size pass through the membrane.

Used for critical applications such as sterilizing and final filtration.

Normal Flow Filtration


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Depth Filtration:

Equipment


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Depth Filtration: Cells and Cellular Debris

Stick to Ceramic Encrusted Fibers in Pads

PROTEIN of INTEREST


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Uses crossflow to reduce build up

of retained components on the

membrane surface

Allows filtration of high fouling

streams and high resolution

Tangential Flow Filtration

vs. Normal Flow Filtration


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Tangential Flow FiltrationTFF

Separation of Protein of Interest

Using TFF with the right cut off filters, the protein of interest

can be separated from other proteins and molecules in the

clarified medium.

HSA has a molecular weight of 69KD. To make sure that the

protein of interest is retained, a 10KD cut-off filter is used.

After we concentrate or ultrafilter our protein, we can

diafilter, adding the phosphate buffer at pH 7.1 that we willuse to equilibrate our affinity column to prepare for affinity

chromatography of HSA.


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How TFF Concentrates and Purifies

a Protein of Interest


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Downstream Processing Equipment

Lab-Scale TFF System Large-Scale TFF System


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Low Pressure Production

Chromatography

The System: Components and Processes

The Media: Affinity, Ion Exchange,Hydrophobic Interaction Chromatography

and Gel Filtration


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LP LC Components

Mixer for Buffers, Filtrate with Protein of

Interest, Cleaning Solutions

Peristaltic Pump

Injector to Inject Small Sample (in our case for

HETP Analysis)

Chromatography Column and Media (Beads)

Conductivity Meter

UV Detector


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Peristaltic Pump

Creates a gentle

squeezing action tomove fluid through

flexible tubing.


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Liquid Column Chromatography

A C i l LP LC


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Lonza, Portsmouth, NH

A Commercial LP LC

Chromatography Column


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Downstream Processing Equipment

Lab Scale

Chromatography System

Large Scale

Chromatography System


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Overview of LP LC Chromatography The molecules of interest, in our case proteins, are adsorbed or

stuck to beads packed in the column. We are interested in the

equilibrium between protein free in solution and protein boundto the column. The higher the affinity of a protein for the bead

the more protein will be bound to the column at any given time.

Proteins with a high affinity travel slowly through the column

because they are stuck a significant portion of the time.

Molecules with a lower affinity will not stick as often and willelute more quickly. We can change the relative affinity of the

protein for the column (retention time) and mobile phase by

changing the mobile phase (the buffer). Hence the difference

between loading buffers and elution buffers. This is how proteins

are separated.

The most common type of adsorption chromatography is ion

exchange chromatography. The others used in commercial

biopharmaceutical production are affinity, hydrophobic

interaction and gel filtration.


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Liquid Chromatography

Protein solution is applied to acolumn

Column filled with matrix (stationary

phase) + liquid phase (mobile phase)

Proteins separated based on

differing affinity for the stationary

and mobile phases

1 2 3 4


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Column Chromatography

Separates molecules by their chemical andphysical differences

Most common types:

Size exclusion (Gel filtration): separates bymolecular weight

Ion exchange: separates by charge

Affinity chromatography: specific binding

Hydrophobic Interaction: separates by

hydrophobic/hydrophilic characteristics


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Biopharmaceutical Production Overview

Typical Process Flow

(Feed 2)

(Feed 3)

(Feed 4)

Chrom 1Chrom 3

Cryo-preservation

Concentration /

Diafiltration

Centrifuge

Viral Removal

Filtration

(Feed1)Inoculum ExpansionAmpoule Thaw

Chrom 2


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What Will Change During Scale-up?Process Development Considerations

Utility requirements

Water requirement

Cleaning/Sanitizing solution requirements

Buffer prep

Number of steps in cell culture scale up

Harvest techniques

Column packing; distribution of introduced liquid atlarge columns

Equipmentbubble trap Automation of process

Data collection

Sample load


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How to survive when scale up process?

Understanding the physics, chemistry and biology of the

chromatographic system and the binding of the protein of

interest to the chromatographic matrix or beads (Science)

Understanding the design and operation of chromatography

components and of the chromatographic process (Technology

and Engineering).

Understanding the calculations needed to run thechromatographic system (column volume) and the

measurements on chromatograms needed to calculate the

HETP, number of theoretical plates, retention time, and

resolution (Mathematics).


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Actual BioLogic System

Complex System

Not easy to see

interaction of components

Students use virtual

system to prepare to use

actual system

Use virtual system for

BIOMANonline

System same as industrial

chromatography skid


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Further reading

G Rao.2007. Introduction to Biochemical

Engineering , Chapter 17

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3 Downstream Process Farmasi

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