Review Article

Towards the Era of Biologics and Biosimilars, a Case of Insulin

Suntara Eakanunkul
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University

Recombinant human insulin is the first protein
therapeutic produced by recombinant DNA technology.
Currently, many patents of insulin analogs start to
expire. The intended-copy recombinant human insulins
manufactured from various manufacturers around the
world have entered the market in Thailand, whereas the
regulatory standard in medicine registration varies widely
between countries. Therefore, the basic concept on protein
therapeutics, biosimilars and insulin quality considerations
will be very useful for physicians, pharmacists, healthcare
professionals to help them evaluate and select high quality
recombinant human insulins and biological products for
hospital formulary.
Keywords: Biologics, biosimilars, biosimilar insulins,
insulin(s), product quality

2558; 30 (6): 630-640 Srinagarind Med J 2015; 30 (6): 630-640

630

2558; 30 (6) Srinagarind Med J 2015; 30 (6)

(biologics or biological products)

5 (protein
therapeutics)

3
erythropoietin, granulocyte
colony-stimulating factors, growth hormone, insulins,
monoclonal antibodies

Suntara Eakanunkul

(recombinant DNA
technology, rDNA)3,4

(immunogenicity)

-
5-7 ( 1)

1 8

(Da)
Heterogeneity
(stability)

1,000-150,000
< 1,000

(variability) 5,9

(heterogeneity)


(batch consistency)
4

Cloning

Protein production

(bioreactor)



Protein purification

2558; 30 (6) Srinagarind Med J 2015; 30 (6)

631

Towards the era of biologics and biosimilars, a case of insulin

.. 1921

Protein formulation

hypersensitivity
reactions

1921

(excipients)

..
hypersensitivity reactions

.. 1978

.. 1982
.. 1978

genetic engineering
.. 1982
(process-related impurities) host cell DNA host genetic engineering
14, 15
cell proteins
14, 15

5,808 Da Isoelectric point (pI)

5.4
2
5,808 Da Isoelectric point

5.4

2 A

high molecular
weight
proteins B (pI)

A
21

30

A
(HMWPs) protein aggregates product-related 21 B
10,11
30

(folding)

impurities

(folding)

alpha helix

A
A
B
alpha helix

helix

12,13

helix
betasheet

2
A
B

beta
sheet

2
(insulin
monomer)

(interaction)

(interaction)

(insulin monomer)

(insulin dimer)

(insulin dimer) B
betaB sheet

beta sheet

(hydrophobic interaction)
(hydrophobic
interaction)

Frederick Banting (insulin hexamer)16
16
Charles

Best

(insulin hexamer)

( 1)
( 1)

(a)

(b)

(c)

1 (a) (b)
(PDB:
The PyMOL Molecular Graphics System,
1 (a) (c)

(b) 4EWZ

(c)
Version 1.7.4 Schrdinger, LLC.)

(PDB: 4EWZ The PyMOL Molecular Graphics System, Version 1.7.4 Schrdinger, LLC.)

2558; 30 (6) Srinagarind Med J 2015; 30 (6)

632
D:\Bow\.\ 26 ..58 6\\ 30 6\08-64_Review_.docx

3



regular insulin intermediate-
acting insulin .. 1936 Hans
Christian Hagedorn
protamine
pI 7.0

neutral
protamine hagedorn insulin (NPH insulin)17


rapid-acting insulin .. 1996
genetic engineering
B
( 2)

Suntara Eakanunkul

regular insulin insulin lispro18, insulin aspart19
insulin glulisine20


2
basal insulin
long-acting insulin insulin glargine
arginine 2 B
asparagine 21 A glycine
deamidation A21
arginine pI 5.4 6.7
pH 4.0
pH
7.0 pI
microprecipitates

15, 21 ( 2)

2 insulin analogs22

Rapid-acting
Insulin aspart

Pro28 Asp

Insulin glulisine

Insulin lispro

Asn3 Lys
Lys29 Glu
Pro28 Lys
Lys29 Pro

Long-acting
Insulin glargine Asn21 Gly 2 Arg Thr30

pH

7.2-7.4
dimer interface
self-association 7.2-7.4
zinc
7.2-7.4
IGF-1
pI precipitation

4.0

European Medicines Agency (EMA)

2558; 30 (6) Srinagarind Med J 2015; 30 (6)

633

Towards the era of biologics and biosimilars, a case of insulin

.. 2005
EMA
(totality of evidence)
(quality aspects) (non-clinical
studies) (clinical studies)
(comparability exercise)
23

(risk management plan, RMP)

24

EMA (specific guidelines)
erythropoietin,
granulocyte-colony stimulating factors, growth hormone,
insulins monoclonal antibodies
EMA ( 3)

3 EMA25

process-related substances
desamido forms, glycosylated forms other forms
insulin receptor IGF-1 receptor (in vitro studies)
(biological tests)
(toxicity tests)
(in vivo studies)
(hypoglycemia)
single dose cross-over
double blind hyperinsulinaemic euglycaemic clamp studies
1
1
6 1



(.)

634

255626

2558; 30 (6) Srinagarind Med J 2015; 30 (6)

Suntara Eakanunkul

.
.
erythropoietin

( 2558)

erythropoietins
non-innovator biologics
(stand-alone registration)

27

33,34

35

(automatic substitution)36

Naming and traceability
(international
nonproprietary name, INN)

INN



(immunogenicity)
36
(interchangeability)

(naming and traceability)
(traceability)

Immunogenicity

non-innovator biologic

(substitution)
pure red cell aplasia

erythropoietin28




(glycosylation)
(brand name)36

(batch number)
protein

aggregates29,30

(batch to batch variability)31
(medication error)


hypersensitivity reactions 32
(pharmacovigilance)

Interchangeability

(switching)

(therapeutic equivalent)

Finnish Medicines Agency

Dutch Medicines Evaluation Board

2558; 30 (6) Srinagarind Med J 2015; 30 (6)

635

Towards the era of biologics and biosimilars, a case of insulin

( 4)
4 37

Insulin
Zinc* Tween20**
m-cresol* phenol*
Protamine sulphate***
Dibasic sodium phosphate sodium acetate

(stabilizer)

-
Glycerin mannitol sodium chloride
Isotonic agent
NaOH, HCl
-
* ** insulin glulisine *** NPH insulin

(certificate of
analysis, COA)
COA
.

COA
38
(finished
products)
39

(Identification)
peptide mapping, N-terminal sequencing,
immunoblotting, isoelectric focusing (IEF), HPLC

(Assay)

HPLC, immunoassay, biomimetic assays
(animal model assays, cell culture-based bioassays)

(Impurity)

HMWPs,

636

desamido insulin other related insulins

HPLC, size-exclusion chromatography (SEC)
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE)

(Safety)
(sterility)
bacterial endotoxins

(Miscellaneous tests)
pH (zinc)



COA

COA
British Pharmacopoeia (BP), United
States Pharmacopeia (USP)

(specification) (acceptance criteria)
regular insulin USP 201539
BP 201540 ( 5)

2558; 30 (6) Srinagarind Med J 2015; 30 (6)

Suntara Eakanunkul

5 regular insulin39, 40
Requirements
Definition
Identification
Assay
Impurities
HMWPs
Related proteins

pH
Zinc
Endotoxin
Preservative
Sterility
Particulate matter(1)
Particulate matter(2)

Specification of regular insulin

USP 2015
Isotonic sterile solution of insulin human in
water for injection
The retention time of the major peak in
the chromatogram
95.0-105.0%

BP 2015
Soluble insulin injection is a neutral solution
of human insulin
The position of the insulin peak in
the chromatogram
90.0-110.0%

Not more than 1.7%

Not more than 2.0%
Content of A-21 desamido and other insulin A-21 desamido is not greater than 5.0%
related compounds is not more than 2.0% and the sum of any other peaks is not
greater than 6.0%
7.0-7.8
6.9-7.8
10-40 ug/100 IU
Not more than 40.0 ug/100 IU
Not more than 80 IU/100 IU
Less than 80 IU/100 IU
Meets the requirements
Meets the requirements
Meets the requirements
Meets the requirements
10 um: not exceed 6000/container
10 um: not exceed 6000/container
25 um: not exceed 600/container
25 um: not exceed 600/container
10 um: not exceed 3000/container
10 um: not exceed 3000/container
25 um: not exceed 300/container
25 um: not exceed 300/container

COA

COA

2558; 30 (6) Srinagarind Med J 2015; 30 (6)

637

Towards the era of biologics and biosimilars, a case of insulin

COA


(long-term stability study)
(in-use stability study)

HMWPs


HMWPs

11


(GMP)

(certificate of pharmaceutical products)

(raw materials)

41

non-innovator
biologics
.

90
42

638

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