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Review of Product Biosimilar Insulin in Indonesia

Angelica Rivera Santoso (148114050); Erica Kusuma Rahayu S. (148114093); Satriavi


Yanuar Deni (148114104); Gabriella Yuliani A. P. (148114110); Petrus Damiani Tosan A.
(148114111); Daniel Lintang Adhi A. (148114115)

1. Introduction
Diabetes mellitus (DM) is a group of metabolic diseases of hyperglycemia resulting
from defects in insulin secretion and/or insulin action. Based on data from the IDF in
2015, the prevalence of diabetes in Indonesia reached 6.2%. Insulin is produced in beta
cells located in the Langerhans islets of the pancreas. Insulin regulates the metabolism of
carbohydrates and fats to maintain blood glucose levels by elevating the uptake of glucose
from the blood to muscles and fatty tissue Human insulin is composed of two polypeptide
chains, the A- and B-chains, which are linked together by disulfide bonds; it has a
molecular mass of 5,808 Daltons, consisting of 51 amino acids. Insulin was originally
extracted from bovine and porcine pancreata for use in patients with diabetes. Nowadays,
biosynthetic human insulin is manufactured by recombinant DNA technology, which has
led to the availability of insulin analogs such as glargine insulin. The production of
biosimilar insulins has the potential to reduce health care costs, expand market
competition, and increase access to insulin treatment among people with type 1 and type 2
diabetes mellitus.
2. Discussion
Cell Development
Recombinant insulin has a complex structure and variations in the manufacturing process
can occur at several stages during the process. A genetically modified host organism
(generally Escherichia coli or a yeast such as Saccharomyces cerevisiae) is used to
synthesize the precursor protein, which subsequently undergoes proteolytic cleavage to
produce active insulin. E. coli is the most widely used host for producing recombinant
proteins and for metabolic engineering, because it is easy to handle and incurs less
incubation costs. In addition, E. coli genes can be easily manipulated. High cell density
fermentation of recombinant E. coli is an effective process for enhancing the final
concentration and production of the protein product. In the high cell density fermentation,
cells are placed in adverse conditions such as nutrient depletion, exhaustion of dissolved
oxygen, elevated osmotic pressure, and by-product formation. To resolve these problems,
the fed-batch fermentation has applied feeding strategies, including constant feeding,
exponential feeding, and indirect feeding such as pH-stat and DO-stat.
Manufacturing Process of Biosimilar Insulin Glargine
Host cell generation
The pPT-GI vector was constructed to express the prepeptide fusion glargine insulin in
E. coli. The coding region for glargine insulin was amplified with human insulin cDNA by using the polymerase chain reaction (PCR). Primer sequences are added. PCR
products were cleaved with restriction enzymes NdeI (Elpis Biotech, Korea) and XhoI
(Elpis Biotech, Korea) and then electrophoresed on 1% agarose gel to isolate a gene
segment of about 300 bp. The isolated cDNAs were subcloned between the NdeI and
XhoI sites of the pPT vector. The constructed pPT-GI vectors were transformed into E.
coli.
Cultivation

The transformed E. coli cells containing pPT-GI vector were cultured at 37C for 19 h
in 10 ml of Luria-Bertani (LB) medium supplemented with 50 g/ml ampicillin. The
cultured cells were inoculated into 1 L of fermentation medium (tryptone 10 g/l, yeast
extract 5 g/l, NaCl 5 g/l, K 2HPO4 5 g/l) in an aerated 2.5 L fermentor. After 12 h culture
at 25C, the temperature was raised to 30C. The fermentation temperature was then
raised from 30C by about 2.3C every 2 h when the absorbance was at least 10 and
finally E. coli cells were cultured at 37C. During the fermentation, cells were fed
glucose and NH4OH as nutrients and to maintain the pH at 6.9, and pO 2 was kept at
30%.
Isolation and Purification
The cells were harvested by centrifugation at 20,000 g for 30 min. The cells were
resuspended in 20 ml of resuspension. Buffer per 1 g of cell weight. Resuspended cells
were disrupted by sonication and centrifuged at 20,000 g, 4C for 30 min to collect
inclusion bodies. The inclusion bodies were collected, and soluble proteins and some of
the cell debris were removed by centrifugation. After centrifugation, the supernatant
was discarded and the precipitate was collected. The collected inclusion bodies
containing prepeptide fusion glargine insulin were washed with 30 ml of inclusion body
washing solution per 1 g of precipitate, and the washed inclusion bodies were collected
by centrifugation at 20,000 g, 4C for 30 min. Lastly, the inclusion bodies were
washed with 30 ml of deionized water per 1 g of precipitate and collected by
centrifugation at 20,000 g, 4C for 30 min.
Folding and Enzymatic Cleavage
The washed inclusion body was solubilized with solubilization buffer solution. The
inclusion body solution was diluted with refolding buffer solution to a final protein
concentration of 0.5 mg/ml. The refolding buffer containing inclusion body was
incubated in 0.1 mM -mercaptoethanol at 4C for 24, 48, 72, and 96 h. After the
refolding reaction, the refolding solution was adjusted to pH 4.5 with 5 N HCl and
centrifuged at 20,000 g, 4C for 30 min. The supernatant containing the refolded
peptide fusion glargine was collected and the precipitate was removed.
Borate (20 mM) was added to the refolded peptide fusion glargine solution. The pH of
the solution was adjusted to 8.5, and citraconic anhydride was added (4.26 g per 1 g
protein). The solution was incubated with stirring at 25C. After 2 h, 9 units of trypsin
was added per 1 mg protein and the solution was incubated at 25C for 5 h. The pH of
the enzyme-reacted solution was then adjusted to 8.5 with 10 N NaOH. For
deacylation, the acidity of the glargine insulin solution was changed to pH 2.5 by
adding glacial acetic acid and then the solution was incubated at 25C for 5 h.
Prepurification and consentration step
Zinc chloride solution (18%) was added to the converted glargine insulin to a final
concentration of 0.1%. The glargine insulin solution was adjusted to pH 6.1 and
incubated at 4C for 16 h and then centrifuged at 20,000 g for 30 min. The supernatant
was removed and the precipitate containing glargine insulin was collected. The pellet
containing human glargine insulin was resolved with sample buffer (7 M urea, 0.25 M
acetic acid, pH 2.5) for ion-exchange chromatography.
Cation-Exchange Chromatography
The protein solution was loaded at a flow rate of 1 ml/min on a 50 ml SP Sepharose
column equilibrated at a flow rate of 5 ml/min with 10 CV (column volume)

equilibration buffer (7 M urea and 0.25 M acetic acid, pH 2.5). The column was washed
at a flow rate of 5 ml/min with 10 CV elution buffer A (7M urea and 0.25M acetic acid,
pH 2.5), and then bound proteins were eluted at a flow rate of 5 ml/min with 6 CV
elution buffers A and B (7 M urea, 0.25 M acetic acid, and 1 M sodium chloride, pH
2.5) by application of a linear gradient (0-1M NaCl). The eluent was monitored at 280
nm and each peak was collected in fraction tubes.
Final step
The recombinant protein then needs further purification and concentration through
crystallization and chromatography methods. This is followed by crystallization and/or
lyophilization, as well as formulation into a product with clinical utility.
Regulation
Indonesian product registration is regulated by the National Agency of Drug and Food
Control (NA-DFC)/BPOM RI, which is responsible for evaluating the efficacy, safety,
risk, quality, and health demands of the Indonesian population. As a result of Indonesias
strict regulations on pharmaceutical product registration regulations, the majority of drugs
approved must be domestically manufactured. Foreign companies should occupy their
own production plan or partner with a local company.
Registrants must submit application documents that can include the drug master file, a
manufacturing license, GMP certificate and manufacturing site master file. Application
submissions should be in compliance with ASEAN Common Technical Documents
(ACTD). ASEAN standards for GMP, clinical studies, stability studies, etc. should be
followed. The timeline for drug registration is typically between 1-3 years.
Biotech market in Indonesia is still relatively small but keeps growing. Patents for
biopharmaceuticals with market volume worth about 1 billion dollars will expire soon. For
example: Lantuss patent (Insulin glargin) which is manufactured by Sanofi-Aventis has
already expired in 2015 causing many biosimilar manufacturers compete to produce its
biosimilar. For example: In December 2015, Eli Lilly Company and Boehringer Ingelheim
Pharmaceuticals, Inc. Announced that the US FDA granted approval for Basaglar (Lantus
Biosimilar) which is launched in the US in December 2016. There is also MK-1293
(Lantus Biosimilar) from Merck has been reported reach its test to phase III test. It shows
us how Insulin Biosimilars develop quickly after Lantus patents expired. For now,
Indonesia hasnt produce Insulin Biosimilar yet but some companies like Phaparos,
Kalbe, Kimia Farma has started to establish biosimilar production (especially Insulin
Biosimilar) by making cooperation with Korea, Indian, China, and Cuba.
The principal of regulation biosimilars products should demonstrate similarity on
quality, safety and efficacy to Reference Biotherapeutics Product (RBP). Indonesia
guidelines on biosimilars, mostly refers to WHO guidelines and also considered other
established biosimilar guidelines. Evaluation of biosimilars product should acquire the
comparability requirement with the reference product, that is quality, non-clinical, and
clinical comparability study.
In 2012, the Indonesians guideline/regulation on biosimilar development still on draft.
Then the regulatory of biosimilar product in Indonesia finally authorized in PERATURAN
KEPALA BADAN POM RI NOMOR 17 TAHUN 2015 TENTANG PEDOMAN
PENILAIAN PRODUK BIOSIMILAR. The existence of these regulation is to respond the
biotechnological developments in Indonesia and the world over the last 10 years. The
development of biosimilar products isnt like as synthetic chemical products which if the
patent had expired, generic product would appear to have same properties and chemical
structure to innovator. Because of its complexity and made using living cells that are

intrinsically diversity, guarantee safety, quality and efficacy are indispensable since the
stages of production until the circulated of that product in Indonesia.
This document is intended for:
1. Explain the concept of biosimilar products and general principles biosimilar product
registration.
2. Provide guidance on biosimilar product registration requirements in Indonesia.
3. Provide guidance biosimilar product evaluation.
This guideline does not apply to vaccines, products derived from blood/plasma,
recombinant blood products and other biological products (eg. products for gene therapy).
In order to be marketed biosimilar product must meet the general and specific
requirements by the guidelines. Generally requirements include biosimilar product
registration, comparability studies biosimilar product with the innovator product and
comparative product-setting arrangements for products biosmilar. Then for the specific
requirements include: (a).Quality evaluation; (B).Non-clinical evaluation; (C).Clinics
evaluation; and (d).Farmakovigilans.
a. Quality evaluation
Contain an assessment of the manufacturing process, characterization,
specifications, technical analysis, and product stability.
b. Non-clinical evaluation
Contains general considerations such as the level of similarity and special
considerations that include pharmacodynamics, pharmacokinetic and toxicology
test through studies in vitro and in vivo.
c. Preclinical evaluation
Is a clinical comparability studies consisting of pharmacokinetic studies,
pharmacodynamic and clinical trials (efficacy studies) to identidikasi if found any
differences between the biosimilar product to the product comparison. Biosimilar
product must pass pharmacokinetic studies, pharmacodynamic test, study PK / PD
confirmation, study the efficacy, safety, imunogenesitas and extrapolation of
efficacy and safety data.
d. Farmakovigilans
Is a study for the discovery, assessment, understanding and prevention of adverse
effects or any product related issues