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 Ocriplasmin (Jetrea) adalah bentuk terpotong
rekombinan dari plasmin manusia dengan berat
molekul 27 kDa. Protease rekombinan yang
merupakan komponen dari permukaan vitreoretinal

Retinal Physician : 9,2012

Manufacture
The recombinant protein ocriplasmin is produced in a
methylotrophic yeast (Pichia pastoris)
The manufacturing process comprises several stages:
*The upstream process involves inoculum preparation , seed
fermentation, production fermentation and dilution.
*The downstream process involves purification of
microplasminogen by chromatography. Following activation
ocriplasmin is further purified before concentration The
purified ocriplasmin is formulated, diluted and then filtered
before final filtration to ensure microbiological quality,
aliquoted in bottles and stored frozen Master Cell Bank and
Work Cell Bank
Preparation and purification
microplasmin
A catalytically active, human microplasmin
was produced by incubation of [Lys]plasmin in buffer at pH
11.0 for up to 12 hr.
The microplasmin was purified by affinity
chromatography that used lysine-Sepharose and soybean trypsin
inhibitor-Sepharose columns.
It is homogeneous and pure by electrophoretic analysis in
NaDodSO4/polyacrylamide gels and by gel filtration on a
Superose 12 column

The molecularweight of the microplasmin determined by

NaDodSO4 gelelectrophoresis is 29,000 and 26,500 under
reducing condition
Preparation and purification
microplasmin
Material and Methods
Protein : native human plasminogen were separated by affinity
chromatography on lysine-Sepharose columns
Reagents. Urokinase, soybean trypsin inhibitor, and the tripeptide substrate
Protein and Enzyme Concentration. The active site concentration
of plasmin or microplasmin was determined by the p-nitrophenyl-p'-
guanidinobenzoate burst titration of Chaseand Shaw

Preparation of Protein-Substituted Sepharoses. Soybean

trypsin inhibitor (40 mg) or urokinase (45,000 units) was
Coupled to CNBr-activated Sepharose 4B (2 g) in 0.1 M
NaHCO3/0.5 M NaCl, pH 8.3. The gel was washed repeatedly
with 100 ml of 0.1 M acetic acid/1 M NaCl, pH 4.0,
alternating five times with 100 ml of 0.1 M sodium borate
buffer, pH 8.5, and stored in 0.1 M sodium phosphate buffer,
pH 8.0.
Preparation and purification
microplasmin
Preparation of Urokinase-Free Human [Lys]Plasmin. Urokinase-
free human plasmin was prepared by activation of
human plasminogen with Sepharose-bound urokinase. One
milliliter of plasminogen (20 mg/ml) in 0.05 M sodium
phosphate/0.02 M L-lysine/0.1 M NaCl/0.001 M EDTA, pH
7.0, was incubated with 0.3 ml of packed gel of urokinase substituted
Sepharose at 30C in a reaction vial (Pierce), with
slow stirring. When maximum plasmin activity was attained
(-3 hr) the activation mixture was forced through a tight
glass-wool plug at the end of a 3-ml plastic syringe by
centrifugation. In all cases at least 80% active plasmin was
obtained as determined by p-nitrophenyl p'-guanidinobenzoate
titration
 NaDodSO4/Polyacrylamide Gel Electrophoresis and
amidolytic activity

The unadsorbed protein fractions contained

protein with Mr 29,000-30,000 (Fig. 4, lane d). The protein
in this peak had no amidolytic activity. Material was eluted
from the inhibitor column with 0.1 M acetic acid, and a
catalytically active protein was recovered. The recovered
enzyme had 70 10% of the amidolytic activity of the
original plasmin.
A peptide chain of Mr 26,500 was observed in this
protein fraction by NaDodSO4 gel electrophoresis analysis
after reduction with mercaptoethanol (fig. 4, lane e). The
purified enzyme also showed only one protein peak on
Superose 12 gel filtration analysis (Fig. 5). This purified,
enzymatically active fragment of plasmin is named
microplasmin

Wu et al, 1987
Karekterisasi
A series of biophysical and analytical characterization assays
were performed to provide details of the structural and chemical
properties of the protein. Parameters including protein sequence
and disulphide bond formation, secondary structure and higher
order conformation were characterised. Size properties,
including the presence of cleaved or truncated variants and
aggregates were determined.
Ocriplasmin was shown to be non-glycosylated. In addition, the
presence of process-related impurities has been determined.
Mass spectrometry and N-terminal sequencing were used for the
structural determination. The expected primary structure could
be confirmed for the main peak of the various analytical
chromatography methods
Karekterisasi
Amidolytic Activities: The enzymatic activity of each
plasmin preparation was measured with the peptide
substrate, NH2-D-Val-Leu-Lys-p-nitroanilide (S-2251), at
37C and in 50 mM Tris-HCl, pH 7.4/0.1 M NaCl. The
substrateconcentration was varied between 0.2 and 4 Km.
The initial rate and substrate concentration data were
analyzed on aLineweaver-Burk plot.
microplasmin is slightly more effective than [Lysiplasmin in
the hydrolysis of NH2-Val-Leu-Lys-p'-nitroanilide.
Active substance
Ocriplasmin is a protein of 249 amino acid residues and
consists of two peptide chains. The first peptide is 19 amino
acid residues long and the second is 230 amino acid
residues long (the peptide bond between the 19th and 20th
amino acid, The peptides are linked together by two
disulfide bonds.
Ocriplasmin is capable of cleaving many proteins, for
example fibronectin, fibrinogen, collagen, laminin and
gelatin. The ocriplasmin activity measured with
physiological substrates like fibronectin, fibrinogen,
collagen, laminin and gelatin showed comparable results
with the proteolytic activity of human plasmin
 Ocriplasmin belongs to the serine protease family and
its intended physiological action is to cleave proteins
present in vitreous and vitreoretinal interfaces.
Ocriplasmins activity is pH dependent; at a neutral
pH, it has ahigh lytic activity. The biological activity of
ocriplasmin in vitreous isestimated to be between 16
and 42 days; variation is thought to be dependent on
endogenous levels of serine protease inhibitors
Ocriplasmin is much more stable and is easier to
prepare, compared with plasmin.
Desain produk dan formulasi
DRUG FORMULATION
The drug product is a sterile, clear and colorless solution with no preservatives
Each vial contains 0.5 mg of ocriplasmin in 0.2 ml solution. After dilution with
0.2 ml of sodium chloride 9 mg/ml (0.9%) solution for injection, 0.1 ml of the
diluted solution contains 0.125 mg ocriplasmin

Incompatibilities
In the absence of compatibility studies, this medicinal product must not be
mixed with other medicinalproducts, other than sterile, preservative-free, non-
buffered diluent sodium chloride 9 mg/ml (0.9%) solution for injection.

Shelf life
18 months

After dilution:
From a microbiological point of view, the product should be used
immediately.
The vial and any unused portion of the diluted solution should be discarded
after single use.
Spesifikasi
The proposed release and stability specifications for Ocriplasmin drug
substance comprise test attributes for:
General Properties: appearance (by visual inspection), pH (Ph. Eur.)
and osmolality
Identity: Size and Epitope and Isoelectric Point;
Purity and Impurities: Molecular Size Variants ( by SDS-PAGE),
Hydrophobic Molecular Variants (by RP-HPLC), Molecular Charge
Variants (by CEX-HPLC) and Molecular Size Variants (by SE-HPLC);
Process related impurities: Residual Host Cell Proteins, Residual Host
Cell DNA ;
Quantity: Protein Concentration (by UV 280 nm);
Potency: Enzyme Kinetic properties;
Other quality characteristics: uniformity of dosage unit Endotoxin (Ph.
Eur.) and Microbiological quality / bioburden (Ph. Eur.)
Handling product and storage
Jetrea is presented in a 2 mL single use Type I glass vials
to be stored at -20 C. A carton secondary pack has been
specifically developed for ocriplasmin drug product to
ensure the vial is secured and kept upright. Due to the
inherent auto-proteolytic activity of Ocriplasmin, Jetrea
must be stored and transported frozen. The supply chain
for ocriplasmin drug product consists of shipment on
dry ice and storage at the distributions sites at -20 C 5
C. Immediately prior to use, the frozen drug product
(200l) is thawed at room temperature and is to be
diluted with an equal volume of 0.9 % (w/v) sodium
chloride to adjust tonicity.
Dosage and administrastion
The recommended dose is 0.125 mg (0.1 mL of the
diluted solution) administered by intravitreal injection
to the affected eye once as a single dose.
Each vial should only be used once and for the
treatment of a single eye. Administration to both eyes
concurrently or within 7 days of the initial injection is
not recommended in order to monitor the post-
injection course including the potential for decreased
vision in the injected eye. Repeated administration in
the same eye is not recommended
Kegunaan
Ocriplasmin, marketed as Jetrea (ThromboGenics)
was approved by the FDA for the treatment of
symptomatic VMA and macular holes<400 m in
diameter in October 2012, and became commercially
available at the end of January 2013 in the United
States
the thrombolytic effect of recombinant human
microplasmin was investigated in animal models of
ischemic stroke, where it gave better results than tPA
(Lapchak et al. 2002; Suzukiet al. 2004a, b). That
induced a shorter bleeding time and decreased
cerebral ischemic damage and improved neurological
dysfunction
 Figure 2. Ocriplasmin used in the treating of vitreomacular adhesion.

Retinal Physician : 9,2012

Daftar rujukan
Quiram PA, 2012. Prospects for Nonsurgical Closure of Macular Holes With Ocriplasmin,
Retinal Physician, 9:3, pp. 46 - 48
Wu LH, Shi GY, and Bender ML, 1987. Preparation dan purification of microplasmin,
Proc. Nati. Acad. Sci: 84. USA pp. 8292-8295
Shi GY,Wu HL. Isolation and characterization of microplasminogen. A low molecular
weight form of plasminogen. J Biol Chem 1988; 263:170715.
Nagai N, Demarsin E, Van Hoef B et al. 2003. Recombinant human microplasmin:
production andpotential therapeutic properties. J Thromb Haemost1:307313
Ma Z, Lu W, Wu S, Chen J, Sun Z, Liu JN .2007 Expression and characterization of
recombinant humanmicro-plasminogen. Biotechnol Lett 29:517523
Suzuki Y, Nagai N, Collen D. Comparative effects of microplasmin and tissue-type
plasminogen activator (tPA) on cerebral hemorrhage in a middlecerebral artery occlusion
model in mice. J Thromb Haemost 2004; 2:16171621.
Lapchak PA, Araujo DM, Pakola S, Song D, Wei J, Zivin JA. Microplasmin: anovel
thrombolytic that improves behavioral outcome after embolic strokesin rabbits. Stroke
2002; 3:22792284.
Weng CY, Ocriplasmin and its Role in theManagement of Vitreo retinal Interface
Disorders
EMA, 2013. Jetrea, Assesment Report