Large Scale Production and Commercialization of Proteins from Recombinant Microorganisms
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Transcript of Large Scale Production and Commercialization of Proteins from Recombinant Microorganisms
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Large Scale Production and Commercialization of Proteins
from Recombinant Microorganisms
Linawati Hardjito, PhD
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BiologyChemistryGenetics
EngineeringStatistics
Economics
Biotechnology
Biodiversity
Process & Products Development
Natural ProductsPharmaceuticalsBiomedical probesNutraceuticalsCosmeticsFood/FeedIndustrial Chemicals
Pollution ControlBioremediationBiofiltrationBioleachingBioabsorption
Aquaculture related product
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Bioprocess Engineering
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Bioreactor 2000 L
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Bioproses proses yang memanfaatkan organisme (khususnya mikroorganisme) untuk mengasilkan produk & proses yang bermanfaat untuk kemidupan manusia dan lingkungannyaSub-disiplin bioteknologi yang berperan merubah penemuan bioteknologi menjadi produk/proses Contoh:
• Produksi rekombinan enzim amilase dengan Saccharomyces cerevisiae dan Pichia pastoris
• Produksi rekombinan selulase, lipase dengan Escherichia coli• Produksi penisilin dengan Penicillium sp• Bioremeidasi tanah terkontaminasi minyak bumi• Produksi biopigmen pycoerythrin dengan mikroalga
Porphyrodium cruentum
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BiologyChemistryGenetics
EngineeringStatistics
Economics
Biotechnology
Marine Biodiversity
Process & Products Development
Natural ProductsPharmaceuticalsBiomedical probesNutraceuticalsCosmeticsFood/FeedIndustrial Chemicals
Pollution ControlBioremediationBiofiltrationBioleachingBioabsorption
Aquaculture related product
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Oganisme/ekstrakterpilih
Pengembangan & Optimasi bioprosesPemilihan media (C,N, mineral,dll)Pemilihan kondisi lingungan fisik & kimia(t,pH,salinas,cahaya,oksigen)
Proses terpilih
Perbaikan genetik Mutasi
Rekayasa genetika
Down stream processing• Pemanenan• pemurnian
Formulasi produk
product
Pengembangan budidaya
FraksinasiPemurnianKarakterisasiStruktur elucidasi
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Basic researchMolecular biology High value added product
Bioprocess Engineering
Bioprocess engineering is the discipline that puts biotechnology to work
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Sifat Unggul BioprocessKondisi reaksi yang ringan/ramah lingkungan
Temperatur & tekanan ruangReaksi bersifat sangat spesifik dan efektif
Enzim/mikroorganisme mengkatalisis reaksi tertentuReaksi yang kompleks dapat diupayakan menjadi satu kesatuan
Beberapa enzim/mikroorganisme bekerja bersama-sama
Menggunakan sumberdaya terbarukanPenerapan rekayasa genetika menungkinan untuk meningkatan produktiftas
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Sifat lemah bioproses
Campuran produk yang kompleks : mikroba, produk, sisa nutrien Mudah terjadi kontaminasi Perlu sterilisasi dan penjagaan kondisi aseptik
Variabilitas dan ketidakstabilan Mutasi alami Mikroorganisme (enzim) tidak bisa bekerja
pada kondisi yang sangat bervariatif
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Produk bioprosesBerdasarkan volume produksi dan nilai jual
Volume besar nilai rendah Volume besar nilai menengah Volume kecil nilai tinggi
Berdasarkan letak produk Intraselular Ekstraselular
Berdasarkan peran dalam metabolisme Metabolit primer Metabolit sekunder
Berdasarkan waktu produksi Growth associated Non-growth associated
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Waktu (jam,hari)
Konsentrasi sel (g/L, OD600nm, jumlah sel/mL)
sel
Growth associated (metabolit primerNon Growth associated (metabolit sekunder)
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Kebutuhan pengembangan bioproses
mikroorganisme nutrien ( sumber karbon, nitrogen, mineral, vitamin, oksigen, dll) Makronutrien Mikronutrien
Kondisi lingkungan yang sesuai (pH, salinitas, temperatur, tekanan)Bioreaktor (tempat kultivasi )
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Penggolongan bioproses
Berdasarkan kebutuhan oksigen Aerobik & anaerobik
Berdasarkan kandungan air dalam campuran substrat : padat & cairBerdasarkan moda operasi : batch, fed-batch, continuousBerdasarkan letak pertumbuhan sel : tersuspensi & tertambat
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Bioreaktor dan moda operasi bioreaktor : bejana, tabung, reaktor yang digunakan untuk menumbuhkan mikroorganismeJenis bioreaktor & metode kultivasi
Batch (curah) : tidak ada penambahan nutrien dan pemanenan produk atau pembuangan limbah selama proses kecuali udara (oksigen) untuk proses aerobik
Fed-batch (curah umpan) : ada penambahan nutrien selama proses tapi tidak ada pemanenan sampai /pembuangan limbah selesai
Continuous (sinambung) : ada penambahan nutrien dan pemanenan /pembuangan limbah selama proses
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Bioreaktor
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Batch proses
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Fin =0Fout =0
F = kecepatan penambahan nutrien (medium) (ml/jam, L/jam)In = masuk, out =keluarFin= Fout = 0
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Keuntungan Sederhana Relatif murah dibandingkan proses
lainnya Proses cepat
Kelemahan Produk inhibisi Substrat inhibisi
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Fed-batch (curah umpan)
Fin = Fml/jam Fout =0
Vo
Vt waktu
Sel & substrat
Substrat (S)
Sel (X)
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Keuntungan Produksi sel pada konsentrasi yang
tinggi (high cell density) Mengurangi substrat inhibisi
Kelemahan Produk inhibisi
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Continuous (sinambung)
Fin = Fml/jam Fout =F
Vt =vo waktu
Sel & substrat
Substrat (S)
Sel (X)
Produk (P)
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Keuntungan Mengurangi inhibisi produk Mengurangi inhibisi substrat Sistem pada kondisi steady state : bagus
untuk menentukan parameter kinetikKelemahan Waktu operasi lama Kemungkinan kontaminasi kompleks
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Monitoring pertumbuhan dan pembentukan produk
Pertumbuhan sel Berat kering (g/L, mg/mL) Kekeruhan ( optical density) :
spektrofotometer Jumlah sel (hemositometer & mikroskop)
Sisa substrat (nutrisi) : g/L, mg/mLPembentukan produk : g/L, unit/mLKondisi lingkungan : pH, T, salinitas, Dissolved Oxygen
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Kinetika Pertumbuhan dan pembentukan produk
Specific growth rate (µ): perrcepatan pertumbuhanµ mengikuti persamaan Monod atau Michaelis Menten
Untuk proses batch (curah) Nutrisi tersedia berlebih (diberikan pada saat awal proses) Nilai Ks kecil µ mendekati µ max
SS
K s
max
SS
K s
max
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Yield (Y) Yx/s : sel yang dihasilkan per substrat yang
dikonsumsi (g sel/ g substrat), Δx/ Δs Yp/s : produk yang dihasilkan per substrat
yang dikonsumsi (g produk/ g substrat), Δp/Δs =Yp/x : produk yang dihasilkan per sel yang
tersedia (g produk/ g sel), Δp/ ΔX
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Kinetika pada proses batch
XdtdX dt
dXX1
dtXdX
tt
t
tx
X
dtXdx
00
tXX
o
t
ln
tXX ot lnln tXX t 0lnln
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tXX t 0lnln
µ
T =waktu
Ln Xt
Ln Xo
XdtdX
XYdt
dS
sx /
XYdtdP
xp /
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Contoh penentuan µWaktu kultivasi
(jam)Sumber karbon laktat
(1 g/L)Sumber karbon laktat
(3g/L)Konsentrasi sel (g/L)
0 0.049 0.0422 0.106 0.1974 0.305 0.2796 0.62 0.6288 1.14 1.032
10 1.23 1.39812 1.248 1.722
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laktat (1 g/L)
0
0.20.40.60.8
11.21.4
0 2 4 6 8 10 12 14
Waktu (jam)
Sel (
g/L)
laktat (1 g/L)
y = 0.403x - 2.971R2 = 0.9911
-4
-3
-2
-1
0
1
0 5 10 15
Waktu (jam)
ln X
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laktat (3 g/L)
0
0.5
1
1.5
2
0 2 4 6 8 10 12 14Waktu (jam)
laktat (3 g/L)
y = 0.3781x - 2.8135R2 = 0.9418
-4
-3
-2
-1
0
1
0 5 10 15
Waktu (jam)
ln X
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Kinetika pada proses continuous (sinambung)
XVFX
dtdX
XY
SSDdtdS
sxi
/
)(
DXXdtdX
F= feed rate (kecepatan umpan, ml/Jam, l/jamV= volume bioreaktor (ml, L, m3)F/V = D : dilutaion rate, nilai konstanD= µX: Sel (g/L)T ; waktu kultivasi (jam, hari)S: Substrat (g/L)P : produk (g/L)
DPXYdtdP
xp /
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waktu
Sel & substrat
Substrat (S)
Sel (X) dx/dt= 0
ds/dt=0
Pada kondisi steady state
DDXXdtdX
,0
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DSS
K s
max
SDDKSDSDKSSKD
s
s
s
max
max
max
DDKS s
max
Sel & substrat
Substrat (S)
Sel (X)
dx/dt= 0
Pada kondisi steady state
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XY
SSDdtdS
sxi
/
)( 0
dtdS
)(
)(
/
/
SSYXD
XY
SSD
isx
sxi
XY
SSDsx
i/
)(
)(/ SSYXD
isx
Sel & substrat
Substrat (S)
Sel (X) dx/dt= 0
Pada kondisi steady state
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DPXYdtdP
xp / DdtdP
,0
XYP xp /
waktu
Sel & substrat
Substrat (S)
Sel (X)
Produk (P)
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Produktifitas bioreaktor sinambung
Sel (X) = X. D (g/L.jam)Sisa substrat (S) = S.D (g/L.jam)Produk (P) = P.D (g/L.jam)
DDKS s
max
SSYX isx (/
xpYXP
/
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Penentuan nilai KsD (1/jam)
S (g/L)
µmax
½ µmax
Ks
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Contoh kasusSuatu bioproses yang memanfaatkan mikroalga (Chlorella sp) digunakan untuk mengolah limbah industri pupuk yang mengnadung ammonium 200 mg/L dengan volume limbah yang dihasilkan adalah 75 m3/jam.
Peraturan pemerintah mengharuskan kadar ammonium dalam air buangan maksimum 5 mg/L. Bila dari hasil percobaan di laboraorium diperoleh data kinetika sebagai berikut : Yx/s =0.1 g sel/g NH4+ dan µmax = 0.5/hari
Tentukan :Berapa ukuran kolam (biorekator) yang harus dibuat supaya tidak terjadi kematian mikroalga (wash out)Berapa konsentrasi mikroalga yang dihasilkan dalam bioreaktorBerapa produktifitas mikroalga yang diperolehBila mikroalga mengnadung vitamin B1 dengan kadar 0.1 ug/gram sel, berapa produktifitas vitamin B1 yang dihasilkan
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Berikut adalah data hasil percobaan di laboratorium untuk pertumbuhan mikroalga Chlorella sp.
Bila mikroalga tersebut akan digunakan untuk mengolah air dari kolam budidaya yang mengandung kadar ammonium 15 mg/L. Bila air tersebut akan diresirkulasi ke kolam dengan persyaratan bahwa kadar ammonium tidak boleh lebih dari 0.1 mg/L dengan kolam pengolah air yang tersedia memilki kapasitas 1000 m3.1.Tentukan berapa flow rate yang diperbolehkan agar air yang dihasilkan dari unit pengolah memenuhi persyaratan yang diinginkan2. Hasil percobaan laboratorium menunjukkan bahwa dari 1 gram ammonium dihasilkan 0.3 g biomasa 3. Berapa maksimum specific growth rate yang dimiliki mikroalga ?
Waktu(hari)
Kepadatan selSel/mL
1 2.5 x 105
2 3.5 x 105
3 7.5 x 105
4 2.15 x 106
5 4.90 x 106
6 5.90 x 106
7 6.05 x 106
8 5.55 x 106
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Komersialisasi
Demand orientedTechnologically feasibleEconomically feasible
Investment, Payback Period, IRR, B/CSocial and environmentally feasible