Biological Products Spec 53

:

:

:

:

11000

: 0-2590-1628 : 0-2590-1634

http://www.phdb.moph.go.th

1 : 2553

: 1,500

3 Biosimilar 10 2 21 17 4 3

... ... ... . .

25532553

1

((BBiioollooggiiccaall PPrroodduuccttss // BBiioollooggiiccss)

4

Biosimilar 5

(Biopharmaceuticals) 5

6

Recombinant-DNA (rDNA) Technology 7

Recombinant Protein 8

Prokaryotic Cell 9

Eukaryotic Cell 10

11

11

12

12

13

/ 13

14

(Identity) 14

16

(Binding Analyses) 23

(Biological Activity) 23

24

28

29

((BBiiooeeqquuiivvaalleennccee))

31

31

32

33

33

34

BBiioossiimmiillaarr 36

BBiioollooggiiccaall pprroodduuccttss

Albumin human.....% injection 37

Enoxaparin sodium......mg /......ml injection 40

Erythropoietin alpha......iu injection or for injection 43

Factor VIIa.....mg injection 46

Factor VIII.....iu injection ( plasma derived) 49

Filgrastim.....mcg injection 52

Immunoglobulin G.....% intravenous injection or for injection 55

Insulin aspart.....iu/ml.....ml injection 58

Insulin glargine.....iu/ml.....ml injection 61

Insulin human.....iu/ml.....ml injection 64

Isophane insulin human.....iu/ml.....ml injection 67

Insulin lispro.....iu/ml.....ml injection 70

Interferon alpha 2.....iu injection 73

Interferon beta 1.....mcg/.....ml injection 76

Peginterferon alpha 2.....mcg /.....ml injection 79

Octreotide.....mg/ml injection 82

Rabies vaccine 85

CChheemmiiccaall ddrruuggss

Cefoperazone sodium.....mg and Sulbactam sodium.....mg for injection 88

Imipenem.....mg and Cilastatin.....mg for injection 91

Meropenem.....g for injection 94

Piperacillin.....g and Tazobactam.....mg for injection 97

101

1

.2543

(Guidelines) (Pattern Specification)

(Specification) 4

1. (Generic Name) ( Dosage Form)

2. (Appearance)

- : ,,,

- :

()

- :

2

- : Labeling

(Expired Date) , (Manufacturing

Date)

3.

general requirements

4.

(Certificate of Analysis )

(Finished Product )

(Specification)

Identification, Label amount, (weigh/unit), Uniformity of dosage

units, Weight variation, Content uniformity, Disintegration time, Dissolution, Related Substances,

Particulate matter, pH, Pyrogens (in vivo) Bacterial endotoxins (in vitro), Leak test, Sterility

test, Microbial limit test, Deliverable volume ,Minimum fill, Water content & Loss on drying,

Chromatographic purity

. 2548

(http://phdb.moph.go.th)

(http://dmsic.moph.go.th)

)Customer enforcement)

(Pattern Specification)

(Specification)

82

3

1.

1.1

1.2

2. Finished product specification (FPS) and Certificate of analysis (COA)

3. Certificate of a Pharmaceutical Product (WHO)

4. BA/BE

5. Specific Rotation / Optical Rotation

6.

7.

8.

9.

2 () .2549

.2552

amino acids

//

22555533

4

((BBiioollooggiiccaall PPrroodduuccttss // BBiioollooggiiccss))

......

/ (Biological Products)

ASEAN HARMONIZATION ? @

? (allergens) (antigens) (vaccines) (hormones)

(cytokines) (enzymes) (stem cells)

(tissues) (human whole blood and plasma

derivatives) (immune sera) (immunoglobulin)

(monoclonal antibodies)

1. (eukaryotic cells)

2. [extraction of substances from

biological tissues including human, animal and plant tissue (allergen)]

3. (recombinant-DNA or rDNA techniques)

4. (hybridoma technique)

5. (propagation of microorganisms in embryo or

animals)

6.

(purity) (potency) (composition) (chemical) (physiochemical) @

(Biopharmaceuticals) (Biological

5

Products) Biologics Biological Products

Biosimilar

Biosimilar ? Follow-on Biologics @

follow-on biologics

impurity

(European Union)

generics

?Similar Biological Medicinal Products@

?comparability@ European

Medicines Agency (EMEA) guidelines

biosimilar

(Biopharmaceuticals)

( biocompatibility )

(identity) (

(primary, secondary, tertiary and quaternary structure)

6

(post-translational modification) (subunit

structure) (purity) (impurity) (quantity) (potency)

(physicochemical properties)

(glycosylation)

rDNA technology, hybridoma technology transformed

continuous cell lines

homogeneity

low molecular weight reference standard

validated

rDNA

7

technology monoclonal technology

transgenic animals transgenic plants, gene therapy

antisense DNA

rDNA technology monoclonal antibody

Recombinant-DNA (rDNA) Technology

Recombinant-DNA (rDNA) technology

(plasmid)

recombinant-DNA (rDNA)

prokaryotes ( ) eukaryotes (

)

E. coli

eukaryotes rDNA prokaryotes

eukaryotes prokaryotes

post-translational modification (glycosylation)

eukaryotes

8

()

rDNA

E.coli E.coli

Recombinant Protein recombinant protein 3

1. Upstream processing

bioreactor

2. Downstream processing

final bulk product

3. Formulation

(finish product)

9

Recombinant Protein

Prokaryotic Cell eukaryotes

E. coli folding

E.coli methionyl derivatives

protease endotoxin

(genetic stability)

DNA restriction mapping

peptide mapping

E.coli yield conformation

potency

(Chromatographic

purification)

upstream downstream upstream downstream formulation

upstream downstream

(Chromatographic

Purification)

/ (Precipitation/

Centrifugation)

(Concentration)

/ (Fermentation)

(Cell Harvest)

10

Eukaryotic Cell eukaryotes

rDNA E.coli

glycoprotein

folding

Chinese Hamster Ovary (CHO) cells

immortal cell lines

Saccharomyces cerevisiae

E.coli

glycoprotein monoclonal antibody

genetic stability , yield

adventitious organism eukaryotes

oncogenic DNA/RNA

E. coli

peptide mapping resolution sensitivity adventitious organisms

mycoplasmas adventitious viruses degree glycosylation

glycoprotein degree

(half-life) (potency)

(antigenicity)

11

(fermentation)

(cell culture) chromatofocusing ,

reversed-phase chromatography , hydrophobic interaction chromatography , charge-transfer

chromatography , size-exclusion chromatography ( molecular sizing ) , ion-exchange

chromatography , affinity chromatography

E. coli protease

protease

monograph European Pharmacopoeia

(EP) British Pharmacopoeia (BP)

intermediary compounds

immunogenicity

current GMP

upstream

( bulk concentrated material) downstream (

purified raw material) intermediate

product

12

prokaryotic cells eukaryotic cells

prokaryotes , ,

karyology, phenotyping

(DNA restriction mapping ) ,

mRNA / plasmid DNA eukaryotes

master cell bank working cell bank adventitious

organisms, karyology, eukaryotic

( ) retroviruses, retroviral activity markers tumorigenicity

stabilizer

solubilizing buffer

lyophylization

deamidation,

aggregation, oxidation proteolysis protease

stabilizer

- (tonicity)

proteins, polyhydric alcohols, amino acids, carbohydrates, bulking agents,

inorganic salts nonionic surfactants lyophilized

monograph

(accelerated stability studies)

(shelf life) protein

conformation Arrhenius

13

(reference standard)

USP, WHO, NIH FDA

(activity units)

(calibrate secondary standard)

/

(numerical limits) (range)

intermediate

Certificate of Analysis (COA)

(physicochemical properties) (biological activity)

(immunochemistry analysis) (purity) (impurities and contaminants)

(quantitative analysis)

International Conference on Harmonization (ICH ; technical guidelines )

(appearance; )

(, circular dichroism (CD), DSC, EPR, MS, IE focusing, isoform

14

pattern, native electrophoresis, NIR, NMR, peptide mapping, 2D electrophoresis, X-ray

diffraction), ( receptor)

() (capillary

electrophoresis, ELISA-HCP, HPIEC, RP-HPLC, HPSEC, LAL test, PCR),

(Kjeldahl analysis, UV absorbance), sterility, pH, osmolarity

COA

monomer multimers

chromatography, electrophoresis

(primary structure)

(comparability)

(Identity)

, post-translational modifications

,

, , , , X-ray

diffraction , NMR

recombinant protein

molecular weight, isoelectric point, ,post-translational modification,

liquid chromatography patterns biomolecular interaction

15

post-translational

modification ( glycosylation, phosphorylation acylation pattern)

immunogenic

allergic

molecular weight electrospray mass

spectrophometry, MALDI-TOF, HPSEC ultrafiltration

isoelectric point (pI) isoelectric focusing (IEF)

polyacrylamide gel capillary electrophoresis (CE-IEF) pI pH

N (N-terminal sequencing) Edman

degradation, C (C-terminal analysis)

(peptide mapping)

RP-HPLC mass spectrophotoscopy

matrix-assisted laser desorption/ionization time-of-

flight (MALDI-TOF)

Fourier transform-infrared/Raman far-UV circular dichroism

near-UV circular dichroism

nuclear magnetic resonance spectroscopy (NMR)

dynamic light scattering

X-ray crystallography

Post-translational modification side group

methionine,

16

deamidation asparagines glutamine, phosphorylation, glycosylation, fatty acid

acylation, farnesylation, sialic acid capping, N-methylation acetylation

modification

RP-HPLC , HPIEC mass spectroscopy

glycosylation

recombinant protein eukaryotic cell lines

glycosyltransferases

(neutral sugars; D-galactose, D-mannose L-fucose), amino sugars (N-acetylglucosamine

N-acetylgalactosamine) acidic sugar sialic acid

oligosaccharides

Protein Sequencing

Protein sequencing

N C

recombinant DNA protein

homogeneity

Peptide Mapping by HPLC

peptide mapping

genetic stability

peptide mapping

endoprotease

Purity

(specific biological activity)

specific biological activity

17

recombinant technology

adventitious

agents specific impurities

Impurities

Contaminants

heterogenous

form form

aggregation

electrophoresis column chromatography (

HPLC)

impurities contaminants

impurities impurities

scrambled form

in-vitro folding

impurities

impurities

impurities

impurities host cell recombinant protein

18

apoptosis lysis impurities endotoxins, viruses, prions, nucleic acid, host

cell lipids, proteins proteolytic enzymes

impurities upstream,

downstream impurities bacteria, yeast, fungi, mycoplasma,

viruses, prions, endotoxins,

impurities des-amido

forms, oxidized forms, scrambled forms, glycosylated forms, cleaved forms, carbamylated

forms, acylated forms polymeric forms

polymeric forms immunogenicity

impurities

Des-Amido Forms amido-

glutamine asparagines amido-

Oxidixed Forms Met, Cys, His, Trp

Tyr oxidation cystinyl

Carbamylated Forms primary amino, sulfhydryl,

carboxyl, phenolic hydroxyl, imidazole phosphate group cyanate

urea

des-Amido forms, oxidixed forms carbamylated forms

HPIEC, RP-HPLC , native PAGE, IEF, MS CE

Aggregates () hydrophobic interaction

aggregates

aggregate aggregates

non reducing agents, 1D-SDS-PAGE, HPSEC, MS CE

hydrophobic aggregates HPSEC

19

Scrambled Forms in-vitro folding HPIEC, RP-HPLC, CE

peptide mapping

Glycosylated Forms glycosylation

HPIEC, RP-HPLC, native PAGE, IEF, MS, peptide mapping CE

Cleaved Forms proteolysis N- C-

HPIEC, RP-HPLC, native PAGE, IEF, MS, peptide mapping CE

Residual Host Cell DNA/Protein

Host Cell DNA/RNA Residual host cell DNA

DNA recovery

DNA DNA

DNA RNA RNA/DNA

RNA retrovirus

Center of Biologics Evaluation and Research (CBER)

10 pg 100 pg

DNA hybridization (dot blot analysis)

DNA

biosensor polymerase chain reaction

(PCR) DNA RNA realtime PCR

20

Host cell protein Host cell protein host

expression system

impurities

process validation finished product

SDS-PAGE, 2D-electrophoresis, western blot immunoassay

Host cell protein generic specific test

generic assays

pattern impurities

specific test assays

host cell protein host cell protein

mammalian cell culture

master seed bank

working cell banks ,,

, ,

pH

Prions transmissible spongiform encephalopathies (TSE)

scrapie , chronic wasting disease , bovine spongiform

21

encephalopathy (BSE) Kuru Creutzfeldt-Jakob disease (CJD)

recombinant protein

prions

( trypsin, serum, transferin, bovine/human serum

albumin, protein supplements, peptones)

semiquantitative assay western blot, capillary immunoelectrophoresis, conformation-

dependent immunoassay dissociation-enhanced,time-resolved fluoroimmunoassay

Microbial agents

Mycoplasma GMP

mycoplasma mycoplasm

Endotoxins endotoxins

(Raw Materials) impurities

prions

(stabilizers)

22

Impurities Contaminants

Impurities Contaminants Impurities Endotoxin Bacterial Endotoxins Test, Pyrogen Test

Host cell proteins SDS-PAGE, Immunoassays

Other protein impurities (media) SDS-PAGE, HPLC, Immunoassays

DNA DNA hybridization, UV spectrophotometry,

Protein binding

Protein mutants Peptide mapping, HPLC, IEF, MS

Formyl methionine Peptide mapping, HPLC, MS

Oxidized methionines Peptide mapping, Amino acid analysis, HPLC,

Edman degradation analysis, MS

Proteolytic Cleavage IEF, SDS-PAGE (reduced), HPLC,

Edman degradation analysis

Aggregated proteins SDS-PAGE, HPSEC

Deamidation IEF, HPLC, MS, Edman degradation analysis

Monoclonal antibodies SDS-PAGE, Immunoassays

Amino acid substitutions Amino acid analysis, Peptide mapping, MS,

Edman degradation analysis

Contaminants

Microbial (bacteria, yeast, fungi) Microbial Limit Tests, Sterility Tests,

Microbiological testing

Mycoplasma DNA-binding Fluorochrome

Viruses (endogenous and adventitious) Cytopathic effect and Hemadsorption (exogenous

virus only), Reverse transcriptase activity,

Murine antibody production

23

(Binding Analyses) binding assay

binding assay

1. ligand** ligand analog binding assay

enzyme-linked immunosorbent assay (ELISA)

epitope

ligand

2. monoclonal antibody binding assay

(Biological Activity)

/ (potency; )

Biomimetic assays animal model assays, cell

culture-based bioassays in-vitro (physicochemical) assays

Animal Model Assays animal facilities , ,

( ) ,

** ligand

24

cell culture-based bioassays in-vitro assay

human growth hormone (somatrem and somatropin)

rat

Cell Culture-Based Bioassays , ( 1-3

),

antiviral activity human -interferon human diploid foreskin cell line

human lung carcinoma cell line (A549)

In-vitro (Physicochemical) Assays (potency test)

(in-vivo)

(in-vitro)

in-vitro in-vivo

in-vitro potency test

,, tissue-type

plasminogen activator (alteplase) in-vitro clot lysis assay

antibody antigen ligand

monoclonal antibody

Protein Content

protein content

protein content

UV spectrophotometry Kjeldahl

reference standards Lowry protein,

biuret, protein content

25

potency

protein content

protein content

UV absorptivity

aromatic amino acid tyrosine, tryptophan phenylalanine

total protein content

des-amido forms, oxidized forms polymeric forms

high performance chromatography

UV

bioassay amino acid

analysis, Kjeldahl analysis, ultraviolet spectrometry high-performance chromatography

Total Protein

total protein

UV spectrophotometry, Kjeldahl assay, bicinchoninic acid (BCA)

assay, biuret assay, Bradford assay, Lowry assay

(total protein)

amino acid analysis, Kjeldahl UV absorbance

ELISA

Immunoassays

Immunoassays

host cell protein

immunoassay

E.coli CHO protein

immunoassays potency monoclonal antibodies

26

immunoassays

radioimmunoassay (RIA) enzyme-linked immunosorbent assays

(ELISAs) ELISA impurity sandwich

ELISA

ELISA (Enzyme Linked Immunosorbent Assay) microtitration plate

Electrophoresis Electrophoretic Assays

homogeneity

denaturation, aggregation, oxidation, deamidation electrophoresis

sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) isoelectric focusing (IEF)

Polyacrylamide Gel Electrophoresis (PAGE)

polyacrylamide gel

sodium dodecyl

sulfate (SDS)

polyacrylamide gel Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis (SDS-PAGE) anionic detergent SDS

denature

27

polyacrylamide

aggregation (

non-denature) denature

sodium dodecyl sulfate (SDS)

immunoblotting

Isoelectric Focusing (IEF) electrophoresis

isoelectric point (pI)

pI phosphorylated forms glycosylated forms

pH

Isoelectric pH (pI) pH

gradient isoelectric pH

homogeneity

pI

protein deamidation pI IEF

glycosylation

sialic acid IEF gel SDS-PAGE

2D-electrophoresis isoelectric focusing

SDS-PAGE pI

isoelectric focusing SDS-PAGE

Capillary Electrophoresis (CE) Capillary Zone Electrophoresis (CZE) capillary polyacrylamide, agarose ampholyte

( isoelectric points)

electro-osmotic flow electrophoresis

(1 10 pL)

28

High-performance Liquid Chromatography (HPLC)

electrophoresis

high-

performance ion-exchange chromatography (HPIEC) , reversed-phase high-

performance liquid chromatography (RP-HPLC) hydrophobicity high-performance

size-exclusion chromatography (HPSEC)

recombinant

hydrophobicity

recombinant protein RP-HPLC, HPIEC, HPSEC

HIC impurities

degradation product

High-performance Ion-exchange Chromatography (HPIEC) impurities oxidized (

methionine) deamidation ( glutamine asparagine)

High-performance Size-exclusion Chromatography (HPSEC)

aggregation fragmentation

Hydrophobic Interaction Chromatography (HIC)

hydrophobicity hydrophobic proteins

29

Prekallikrein Activator (PKA)

human albumin immunoglobulin

prekallilrein activator Blood coagulation factor XIIa fragments

PKA final product

Immunochemical Properties

Efficacy

Efficacy

Potency

(Room Temperature)

?@ 18L25C

(Sensitivity) Limit of detection

(Specificity)

false-positives

30

Sialic acid

Silalic acid N-linked O-linked-Neuraminic acid derivatives

sialic acid

in-process control glycosylation

Normocythemic mice/Polycythemic mice

hematocrit ( normocythemia) hematocrit

(polycythemia)

erythropoietin

1. Crommelin, D.J.A., R.D. Sindelar, and B. Meibohm, Pharmaceutical biotechnology :

fundamentals and applications. 3rd ed. 2008, New York: Informa Healthcare. xiv, 466 p.

2. Gad, S.C., Handbook of pharmaceutical biotechnology. 2007, Hoboken, N.J.: Wiley-

Interscience. xix, 1659 p.

3. Niazi, S., Handbook of biogeneric theraupetic proteins : regulatory, manufacturing, testing,

and patent issues. 2005, Boca Raton: Taylor & Francis. 554 p.

4. Rathore, A.S., R. Mhatre, and Wiley InterScience (Online service), Quality by design for

biopharmaceuticals principles and case studies. 2009, Wiley: Hoboken, N.J. p. xvi, 288 p.,

[16] p. of plates.

5. Walsh, G., Pharmaceutical biotechnology : concepts and applications. 2007, Chichester,

England ; Hoboken, NJ: John Wiley & Sons. xviii, 480 p.

6. Wu-Pong, S. and Y. Rojanasakul, Biopharmaceutical drug design and development. 2nd

ed. 2008, Totowa, N.J.: Humana Press. x, 375 p.

7. The United States Pharmacopoeia XXXIII.

8. The European Pharmacopoeia 6th edition.

31

((BBiiooeeqquuiivvaalleennccee))

......

Bioequivalence

.. 2535

(new chemical entity)

Bioequivalence (BE)

(pharmaceutical equivalence) pharmaceutical alternative

(same molar dose)

(same route of administration) bioavailability

(pharmaceutical equivalence)

chlordiazepoxide

hydrochloride 5 mg capsule

/ /

tetracycline hydrochloride 250 mg capsule

32

pharmaceutical alternative tetracycline phosphate 250 mg capsule quinidine sulfate

200 mg tablet pharmaceutical alternative quinidine sulfate 200 mg capsule

Bioavailability (BA)

100%

100%

100%

(therapeutic equivalence)

Orange Book 30th

edition ) . .2553)

section 505 of the Federal Food, Drug, and Cosmetic Act

1. 2.

3.

4. 5. (good manufacturing practice, GMP)

ASEAN Guidelines for

the Conduct of Bioavailability and Bioequivalence Studies

ASEAN Harmonization 1 2552

33

1 2553

GLP ISO/IEC 17025

pharmaceutical alternative

) ( 12 )

20%

18-24 (

(Cmax , maximal plasma concentration) (AUCt or , area under the

plasma concentration-time curve from administration to last observed concentration at time t or

to infinite time) Cmax AUCt or

log (logarithmic transformation) ANOVA

) ( formulation, period, sequence subject (within sequence) 90%

confidence interval Cmax AUCt or

90% confidence interval

Cmax AUCt or 0.80-1.25

90% confidence interval 0.75 - 1.33

1.

(low therapeutic index/ narrow safety margin)

(therapeutic inequivalence)

2.

34

3. (modified release dosage form)

(transdermal dosage form)

4. (fixed combination products)

5. 6.

1.

(high solubility) (high permeability)

Biopharmaceutical Classification System (BCS) 1 )

( (low therapeutic

index/ narrow safety margin) (rapid

dissolving) (very rapidly dissolving)

(polymorphism)

2. elixir, syrup, tincture reconstituted powder

for oral solution

3.

35

4.

5. (inhalation)

6. preservative, buffer, tonicity

/ 7.

8. /

1. . :

, .. 2552.

2. Drug Control Division, Food and Drug Administration. Guidelines for the conduct of

bioavailability and bioequivalence studies adopted from ASEAN Guidelines for the Conduct

of Bioavailability and Bioequivalence Studies. Nonthaburi; Food and Drug Administration,

2009.

3. US Department of Health and Human Services, Food and Drug Administration, Center for

Drug Evaluation and Research, Office of Pharmaceutical Science, Office of Generic Drugs.

Orange book, 30th edition.(2010). Available at: www.fda.gov/Drugs/DevelopmentApproval

Process/ucm079068.htm (access date: 9 April 2010)

36

BBiioossiimmiillaarr**

1. Enoxaparin sodium ??.. mg / ........ ml injection

2. Erythropoietin alpha ..??.. iu injection or for injection

3. Filgrastim ??.. mcg injection

4. Insulin aspart ??..iu/ml ??.. ml injection

5. Insulin glargine??.. Units /ml ??.. ml injection

6. Insulin human ??.. iu/ml ??.. ml injection

7. Isophane insulin human ??.. iu/ml ??.. ml injection

8. Insulin lispro??..iu/ml ??..ml injection

9. Interferon alpha 2???.. iu injection

10. Interferon beta 1???..mcg/??...ml injection

** Biosimilar EMEA

37

AAllbbuummiinn hhuummaann

.... %% iinnjjeeccttiioonn

Albumin human

.. % injection

1. colloid

2. albumin human ....... g .. ml

3.

4. -

- Sodium

-

1. Identification

- Immunoelectrophoresis

- Ouchterlony test (Double immunodiffusion)

2. pH finished product specification

3. Total protein 94 - 106 %

4. Albumin albumin 95%

5. Haem content finished product specification

6. Prekallikrein activator [PKA] finished product specification

7. Sodium 160 mmol/l 95 -105 %

sodium

8. Sterility sterile

9. Pyrogens finished product specification

10. Aluminium finished product specification

11. Potassium finished product specification

38

12. Heat stabilizers finished product specification

N-acetyl-DL-tryptophan,

caprylic acid /caprylate

heat stabilizer

heat stabilizer heat stabilizer

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1

(finished product specification)

finished product

specification

2.

(GMP)

Certificate of pharmaceutical

products

3.

3.1 (Certification of analysis)

3.2 (Raw material)

3.3

- Anti-HIV-1 , Anti-HIV-2 ,

Hepatitis B Surface antigen, Anti-HCV ( Antibody against Hepatitis C Virus)

39

3.4 NIBSC (National

Institute for Biological Standards and Control), AABB(American Association Blood

bank), PPTA (Plasma Protein Therapeutic Association),

4.

4.1 ..........

5.

5.1 .......... ( / )

5.2

5.3

/

5.4

5.5 cold chain system

6.

6.1 Long Term Stability

6.2 (Lot Release)

7.

Administration set membrane filter

40

EEnnooxxaappaarriinn ssooddiiuumm

.... mmgg // ................ mmll iinnjjeeccttiioonn

Enoxaparin sodium

.. mg / ........ ml injection

1.

2. enoxaparin sodium ... mg/ml ( iu anti-Xa / ml )

3.

4.

1. Identification finished product specification

2.

Anti -Xa activity 90.0 - 110.0 %

3. Anti - IIa activity 20 - 35 iu /mg

4. Ratio of Anti-Xa/Anti-IIa 3.3 - 5.3

5. pH 5.5 -7.5

6. Bacterial endotoxins 0.01 endotoxin units/anti-factor Xa iu

7. Volume in container finished product specification


9. Particulate matter finished product specification

41

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- molecular weight

- free sulfate content 0.12%

4.

4.1 ..........

42

5.

5.1 .......... ( / )

5.2

5.3

/

5.4

6.


43

EErryytthhrrooppooiieettiinn aallpphhaa....

.... iiuu iinnjjeeccttiioonn oorr ffoorr iinnjjeeccttiioonn

Erythropoietin alpha ..

.. iu injection or for injection

1. solution

lyophilized form

2. recombinant erythropoietin alpha .. iu

3. (pre-filled syringe)

4. -

- erythropoietin alpha

1. Erythropoietin identity 1 5 (

) - CZE IEF

- PAGE + immunoblotting

- Peptide mapping / LC

- N-terminal sequence analysis

- HPLC

2. Assay

-Biological assay (Potency) Biological assay

1 3

- In polycythaemic mice (in vivo)

- In normocythaemic mice (in vivo)

- Immunoassay (in vitro)

potency 80 - 125%

44

- HPLC () HPLC erythropoietin

finished product specification


4. Bacterial endotoxins/ Pyrogens finished product specification



7. Extractable volume ( solution) finished product specification

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

45

3.3 - residual host cell DNA residual host cell protein

- sialic acid

- dimer / related substances of higher molecular mass

SEC (Size-Exclusion Chromatography) HPSEC 2%

4.

4.1 ..........

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


46

FFaaccttoorr VVIIIIaa

.. mmgg iinnjjeeccttiioonn

Factor VIIa

. mg injection

1.

2. human coagulation factor VIIa . mg

3.

4.

1. Potency 15 iu of factor VII 1 ml

2. Solubility 10

3. Assay of human coagulation factor VII 80 -125% of the stated potency

4. pH 6.5 - 7.5

5. Heparin ( heparin) heparin

0.5 iu heparin/ iu factor VII

6. Coagulation test ( heparin) coagulation 30

thrombin

7. Water content finished product specification



10. Residual host cell protein/DNA

11. Protein identification

47

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3 - Factor II Not more than 125% of the stated content. The confidence limits

(P=0.95) are not less than 90% and not more than 111% of the

estimated potency

- Factor IX Not more than 125% of the stated content. The confidence limits

(P=0.95) are not less than 80 % and not more than 125% of the

estimated potency

- Factor X Not more than 125% of the stated content. The confidence limits

(P=0.95) are not less than 90% and not more than 111% of the

estimated potency

48


Hepatitis B Surface antigen , Anti-HCV (Antibody against Hepatitis C Virus)

- recombinant-human factor VII residual host

cell DNA residual host cell protein

4.

4.1 ..........

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


6.2 (Clinical Trial) Route of Administration

49

FFaaccttoorr VVIIIIII

.. iiuu iinnjjeeccttiioonn (( ppllaassmmaa ddeerriivveedd))

Factor VIII

.. iu injection

1.

2. human coagulation factor VIII .. iu human plasma

3.

4. -

- human derived factor VIII

1. Specific activity 1 iu AFU factor VIII

(1AFU=1 iu)

2. Factor VIII activity 80 - 160% of stated potency

3. Solubility 10

4. pH 6.5 - 7.5


6. Anti-A haemagglutinins 1 to 64 dilutions do not show agglutination

7. Anti-B haemagglutinins 1 to 64 dilutions do not show agglutination


9. Stabilizer finished product specification

50

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- Anti-HIV-1, Anti-HIV-2 , Hepatitis B Surface antigen , Anti-HCV

( Antibody against Hepatitis C Virus)

4.

4.1 ..........

51

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


52

FFiillggrraassttiimm

.... mmccgg iinnjjeeccttiioonn

Filgrastim

... mcg injection

1.

2. filgrastim ... mcg (... iu) .. ml

3. 4.

1. Identification 1 5

- CZE IEF

- PAGE + immunoblotting


- N-terminal sequence analysis

- HPLC

2. Potency 80 - 125%


4. Bacterial endotoxins 2.5 endotoxin units/mg

5. pH 3.8 - 4.2

6. Extractable volume finished product specification

7. Particulate matter visible particle practically free from particles

Spec.

- stabilizer

53

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3


Hepatitis B Surface antigen, Anti-HCV ( Antibody against Hepatitis C Virus)

- purity : 95% main peak

- impurities with higher molecular masses : 2%

4.

4.1 ..........

54

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


55

IImmmmuunnoogglloobbuulliinn GG

..%% iinnttrraavveennoouuss iinnjjeeccttiioonn oorr ffoorr iinnjjeeccttiioonn

Immunoglobulin G

..% intravenous injection or for injection

1. 1 (slightly opalescent)

2

2. 1 immunoglobulin Gg .ml ....... ml

2 immunoglobulin Gg ml ....... ml

3.

4. -

-

1. Identification

- Immunoelectrophoresis

- Immunodiffusion

2. Anticomplimentary activity 50% (1 CH50 /mg immunoglobulin)

3. Prekallikrein activator [PKA] 35 iu/ml (30g/l immunoglobulin)

4. Anti-A and Anti-B Haemagglutinins 1 to 64 dilutions do not show agglutination

5. Anti-HBsAg 0.5 iu/g immunoglobulin

6. Osmolality 240 mOsmol/kg


8. Anti D antibodies finished product specification

9. Total protein 30 g/l 90-110%

10. Molecular distribution finished product specification

(Monomer + Dimer)

56

11. Protein composition Electrophoresis : Not more than 5% of

protein has a mobility different from that of the

principle band

12. Water ( lyophilized powder) finished product specification



1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- Anti-HIV-1, Anti-HIV-2 , Hepatitis B Surface antigen , Anti-HCV

(Antibody against Hepatitis C Virus)

- anti-D Antibodies

57

3.4 NIBSC (National

Institute for Biological Standards and Control), AABB(American Association Blood

bank), PPTA (Plasma Protein Therapeutic Association),

4.

4.1 ..........

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


6.2 (Lot Release)

7.

Administration set membrane filter

58

IInnssuulliinn aassppaarrtt

....iiuu//mmll

....mmll iinnjjeeccttiioonn

Insulin aspart

..iu/ml

.. ml injection

1.

2. insulin aspartiu/ml ....... ml

3.

4. -

- - (avoid freezing)


2. 90.0-110.0% of labeled amount of insulin aspart

3. pH 7.2 - 7.6

4. Limit of high molecular weight proteins 1.5%

5. Related proteins B28isoAsp insulin aspart 2.5%

5%

6. Total Impurities 3.5%

7. Zinc content 16.3 - 24.5 mcg/ml

8. Preservative finished product specification


10. Bacterial endotoxins 80 endotoxin units/100 insulin aspart units

59

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- recombinant-human insulin residual host cell

DNA residual host cell protein

4.

4.1 ..........

60

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


61

IInnssuulliinn ggllaarrggiinnee

.. iiuu//mmll


Insuline glargine

.. iiuu//mmll

.. ml injection

1.

2. Insulin glargine .. iu /ml ........ ml

3.

4. -



2. 95.0 -105.0 % of labeled amount of insulin glargine

3. pH 3.5 - 4.5


5. Related proteins

Largest 0.5%

Sum 2.0%



8. Zinc content finished product specification


10.Bacterial endotoxins 80 endotoxin units/100 insulin glargine units

62

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3



4.

4.1 ..........

63

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


64

IInnssuulliinn hhuummaann

.... iiuu//mmll

.... mmll iinnjjeeccttiioonn

Insulin human

.. iu/ml

.. ml injection

1.

2. insulin human recombinant DNA

insulin mono-component .. iu/ml ........ ml

3.

4. -



2. 90.0 - 110.0 % of labeled amount of insulin human



5. Zinc content finished product specification

6. Bacterial endotoxins 80 endotoxin units/ 100 insulin human units

7. Limit of high molecular weight proteins finished product specification


9. Extractable volume finished product specification

65

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3



4.

4.1 ..........

66

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


67

IIssoopphhaannee iinnssuulliinn hhuummaann

.... iiuu//mmll


Isophane insulin human

.. iu/ml

.. ml injection

1.

2. zinc insulin human protamine sulfate buffered water for injection

.. iu/ml ........ ml

3.

4. -

- insulin

-

- (avoid freezing)

- (shake carefully before use)


2. 90.0 - 110.0 % of labeled amount of insulin human

3. pH 6.9 - 7.8


5. Zinc content 0.021- 0.04 mg/100 insulin human units

6. Bacterial endotoxins 80 endotoxin units/ 100 insulin human units



9. Related substances

A21 desamido insulin finished product specification

Others finished product specification

10. Insulin in the supernatant 2.5% of total insulin content

68

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3



4.

4.1 ..........

69

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


70

IInnssuulliinn lliisspprroo

....iiuu//mmll


Insulin lispro

..iu/ml

..ml injection

1.

2. Insulin lispro iu/ml ....... ml

3.

4. -


1. Identification

2. 90.0 - 110.0% of labeled amount of insulin lispro

3. pH 7.0 - 7.8




7. Zinc content 14 - 35 mcg/100 USP unit of insulin


9. Bacterial endotoxins 80 endotoxin units/100 insulin lispro units

10 Related substances

A21 desamido insulin finished product specification

Others finished product specification

71

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3



4.

4.1 ..........

72

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


73

IInntteerrffeerroonn aallpphhaa 22

.... iiuu iinnjjeeccttiioonn

Interferon alpha 2

.. iu injection

1.

2. interferon alpha 2 . iu

3.

4. -

- interferon alpha 2a 2b

1. Appearance of the solution finished product specification


- CZE IEF

- PAGE / immunoblotting


- Anti viral activity

- HPLC


4. Osmolality finished product specification


6. Bacterial endotoxins 100 endotoxin units / of interferon

finished product specification

7. 84.0 - 116.0% of labeled amount of interferon

74

8. Purity HPIEC HPSEC finished product specification

/ Impurities oxidised forms, dimers

and high molecular weight substances

9. Anti Viral Assay 80 - 125 % of the stated potency

10. preservative

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- recombinant-human interferon residual host


75

4.

4.1 ..........

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


76

IInntteerrffeerroonn bbeettaa 11

..mmccgg//..


Interferon beta 1

..mcg/

...ml injection

1.

2. interferon beta 1..mcg ........ml

3.

4. -

- interferon beta 1a 1b



- CZE IEF




- HPLC




6. Bacterial endotoxins 100 endotoxin units / mg of interferon

7. 84.0 - 116.0% of labeled amount of interferon


/ Impurities oxidised forms, dimers


77

9. Anti Viral Assay 80 - 125 % of the stated potency

10. Moisture / Water () 3%

11. preservative

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- recombinant-human interferon residual host


4.

4.1 ..........

78

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


79

PPeeggiinntteerrffeerroonn aallpphhaa 22

..

.... mmccgg //


Peginterferon alpha 2... mcg /

.. ml injection

1.

2. peginterferon alpha 2. mcg ....... ml

3.

4. -

- peginterferon alpha 2a 2b



- CZE IEF




- HPLC




6. Bacterial endotoxins 100 endotoxin units/mg of peginterferon

7. 84.0 - 116.0% of labeled amount of peginterferon


/ Impurities oxidised forms , dimers


80

9. Anti Viral Assay 50 - 150% of the stated potency.

10. preservative

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- recombinant-human peginterferon residual

host cell DNA residual host cell protein

4.

4.1 ..........

81

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


82

OOccttrreeoottiiddee

.... mmgg//mmll iinnjjeeccttiioonn

Octreotide

.. mg/ml injection

1.

2. octreotide .. mg ........ ml

3.

4.


2. 90.0 -110.0% of labeled amount of octreotide

3. pH 3.9 - 4.5

4. Volume in container finished product specification



7. Bacterial endotoxins 100 endotoxin units/mg of octreotide

8. Total degradation products 2.0%

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

83

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3 - recombinant-human octreotide residual host


4.

4.1 ..........

5.

5.1 .......... ( / )

5.2

5.3

/

84

5.4


6.


85

RRaabbiieess vvaacccciinnee

Rabies vaccine

1.

2. inactivated vaccine (vero cells primary chick/duck

embryo fibroblast cells human diploid cell) rabies antigen 2.5 iu

3.

4. -

- 2 - 8

1. Potency 2.5 iu/dose



4. pH 7.0 - 7.8

5. Bovine serum albumin 50 ng/dose

6. Pyrogens

Bacterial endotoxins 25 endotoxin units/dose

7. Safety

Spec.

- ID 0.7 iu/0.1 ml

- albumin albumin

86

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- tissue culture residual host cell DNA

residual host cell protein

4.

4.1 ..........

87

5.

5.1 .......... ( / )

5.2

5.3

/

5.4


6.


88

CCeeffooppeerraazzoonnee ssooddiiuumm

mmgg aanndd SSuullbbaaccttaamm ssooddiiuumm

..mmgg ffoorr iinnjjeeccttiioonn

Cefoperazone sodium

mg and Sulbactam sodium

mg for injection

1.

2. cefoperazone sodium cefoperazone .. mg

sulbactam sodium sulbactam .. mg

3.

4.


2.

Cefoperazone 95.0 - 105.0% of labeled amount of cefoperazone

Sulbactam 95.0 - 105.0% of labeled amount of sulbactam

3. pH 4.5 - 6.5

4. Uniformity of dosage units finished product specification


6. Bacterial endotoxins finished product specification


8. Constituted solution finished product specification

9. Water content 4.0 %

10.Related substances finished product specification

Chromatographic purity specific impurity

89

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3 - related substances cefoperazone sodium

any other impurities : 1.5 %

total impurities : 4.5 %

4.

4.1 ..........

90

5.

5.1 .......... ( / )

5.2

5.3

/

5.4

6.

6.1 2 Long Term Stability

6.2

91

IImmiippeenneemm

.... mmgg aanndd CCiillaassttaattiinn

.. mmgg ffoorr iinnjjeeccttiioonn

Imipenem

. mg and Cilastatin

. mg for injection

1.

2. imipenem .. mg cilastatin sodium cilastatin.. mg

3.

4.

1. Identification

Imipenem finished product specification

Cilastatin finished product specification

2.

Imipenem 90.0 -115.0 % of labeled amount of imipenem

Cilastatin 90.0 -115.0 % of labeled amount of cilastatin

3. pH 6.5 - 8.5



6. Bacterial endotoxins 0.17 endotoxin units/mg of imipenem

0.17 endotoxin units/mg of cilastatin


8. Loss on drying 3.5%

92

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

Potency (Raw material)

Imipenem

imipenem : 98.0 J 101.0% of imipenem monohydrate

specific rotation : between +84 and +89

related substances

thienamycin : not more than 1.0%

any other impurities : not more than 0.3%

sum of impurities other than thienamycin : not more than 1.0%

93

Cilastatin

cilastatin : 98.0 -101.5% of cilastatin sodium, calculated on

the anhydrous and solvent free basis

specific rotation : between +41.5 and +44.5

4.

4.1 ..........

5.

5.1 .......... ( / )

5.2

5.3

/

5.4

6.


6.2

94

MMeerrooppeenneemm

...... gg ffoorr iinnjjeeccttiioonn

Meropenem

.. g for injection

1.

2. meropenem trihydrate anhydrous meropenem

meropenem g sodium carbonate

3.

4.


2. 90.0 -120.0% of labeled amount of meropenem

3. pH (1 in 20) 7.3 - 8.3


5. Bacterial endotoxins 0.125 endotoxin units/mg of meropenem



8. Chromatographic impurity

- Impurity with a retention time of about 0.8 %

0.45 relative to meropenem

- Impurity with a retention time of about 0.6 %

1.9 relative to meropenem

9. Loss on drying 9 - 12 %


11. Content of Sodium 80 - 120% of labeled amount of sodium

95

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3 - specific rotation: -17 and -21

- chromatographic purity

at relative RT about 0.45 : 0.3 %

at relative RT about 1.90 : 0.3 %

any other impurities : 0.1 %

sum of all other impurities : 0.3 %

- limit of acetone : 0.05 %

96

4.

4.1 ..........

5.

5.1 .......... ( / )

5.2

5.3

/

5.4

6.


6.2

97

PPiippeerraacciilllliinn

.... gg aanndd TTaazzoobbaaccttaamm

.... mmgg ffoorr iinnjjeeccttiioonn

Piperacillin

.. g and Tazobactam

.. mg for injection

1.

2. piperacillin sodium piperacillin .. g tazobactam sodium

tazobactam.. mg

3.

4.

1. Identification

Piperacillin finished product specification

Tazobactam finished product specification

2.

Piperacillin 90.0 - 120.0 % of labeled amount of piperacillin

Tazobactam 90.0 - 115.0 % of labeled amount of tazobactam




6. Bacterial endotoxins 0.07 endotoxin units/mg of piperacillin




10.Related compounds finished product specification

98

1. (declare)

1.1 (.2 .3 .4 )

1.1.1 ( .2)

1.1.2 ( .3)

1.1.3 ( .4)

1.2 . 1


finished product

specification

2.

(GMP)


products

3.


3.2 (Raw material)

3.3

- specific rotation

: piperacillin : +155 +175

- related compounds

: piperacillin specific related substances

: tazobactam specific related substances

4.

4.1 ..........

99

5.

5.1 .......... ( / )

5.2

5.3

/

5.4

6.


6.2

101

()

99 /2552

----------------------------

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

102

-2-

17. 18. 19. 20. 21. 22. 23. 24.

1.

2. 3.

10 .. 2552

( )

( )

/

(Biological Products / Biologics) (Bioequivalence) Biosimilar* Biological productsAlbumin human .... % injectionEnoxaparin sodium .. mg / ........ ml injectionErythropoietin alpha .... iu injection or for injectionFactor VIIa . mg injectionFactor VIII .. iu injectionFilgrastim ... mcg injectionImmunoglobulin G ..% intravenous injection or for injectionInsulin aspart ..iu/ml .. ml injectionInsuline glargine.. iiu//mll .. ml injectionInsulin human .. iu/ml .. ml injectionIsophane insulin human .. iu/ml .. ml injectionInsulin lispro..iu/ml ..ml injectionInterferon alpha 2.. iu injectionInterferon beta 1..mcg/...ml injectionPeginterferon alpha 2... mcg / .. ml injectionOctreotide .. mg/ml injectionRabies vaccine

Chemical drugsCefoperazone sodium mg and Sulbactam sodium mg for injectionImipenem . mg and Cilastatin . mg for injectionMeropenem .. g for injectionPiperacillin .. g and Tazobactam .. mg for injection

Documents

Biological Products Spec 53