DMF for Heparin Sodium Hebei Changshan Biochemical Pharmaceutical Co., Ltd.

3.2.S.3.2 Impurities

Our company has manufactured heparin sodium for many years, and the impurity profile is

provided here. The impurities are divided into 3 categories, and the detailed information is as

follows:

 Possible impurities arising from the synthesis and the raw materials

Taking into account of the starting material crude heparin, which is derived from porcine

intestines, there are mainly 4 possible impurities (i.e., protein, nucleotidic impurities,

dermatan sulfate, oversulfated chondroitin sulfate) in the starting material, which may be

carried over in the final substance.

Furthermore, there are another 4 possible impurities (i.e., bacterial endotoxins,

microorganisms, residue on ignition and heavy metals) arising from the impurities in the raw

materials.

Residue on ignition

This test is usually used for determining the content of inorganic impurities in an organic

substance. Residue on ignition in the final product are controlled according to the limit

regulated in current U.S.P. (28.0%～41.0%) and the batch analysis results show that the level

of residue on ignition in heparin sodium can meet the specification requirement.

Protein and nucleotidic impurities

These two impurities, which originate from the staring material, may be brought into the

manufacture. However, the hydrolysis procedure by sodium chloride separates a great part of

protein and nucleotide from heparins, then following a series of adsorption, elution and

precipitation, most of the protein and nucleotide can be removed from the manufacturing

process; the oxidation procedure by hydrogen peroxide reduces protein and nucleotide into

low molecular masses which would sequentially be largely removed by membrane filtration.

What’s more, the two impurities are more soluble in ethanol than heparin sodium and it is

easier to precipitate heparin sodium with low concentration of ethanol while the two

impurities are still dissolved in the water-ethanol solution, so the two impurities can be

further removed through those precipitation steps by recovered ethanol or ethanol.

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The content of protein and nucleotidic impurities in heparin sodium is strictly controlled

according to the limit (nucleotide ≤0.1%; protein ≤0.1%) regulated in the monograph of

heparin sodium in current U.S.P. and the batch analysis results show that the level of protein

and nucleotide in heparin sodium can meet the specification requirement.

Oversulfated Chondroitin Sulfate

Oversulfated chondroitin sulfate is the impurity which is artificially added into heparin

sodium. Two methods in the current U.S.P. monograph of heparin sodium can effectively

verify its absence.

The current U.S.P. monograph of heparin sodium specifies:

Oversulfated chondroitin sulfate should be absent. The control method is as follows:

A: Proceed as directed in Identification test A. No features associated with oversulfated

chondroitin sulfate are found between 2.12 and 3.00 ppm.

B: Proceed as directed in Identification test B. No peaks corresponding to oversulfated

chondroitin sulfate should be detected eluting after the heparin peak.

The batch analysis data shows that the oversulfated chondroitin sulfate is absent.

Dermatan sulfate

The current U.S.P. monograph of heparin sodium specifies: The percent galactosamine peak

area of the total hexosamine of the Hydrolyzed sample solution must be NMT 1%. It is a

measure of dermatan sulfate and other galactosamine containing impurities. In this method,

the organic impurity (i.e. dermatan sulfate) is hydrolyzed to galactosamine and thus

effectively measure the dermatan sulfate and other galactosamine containing impurities. The

batch analysis data shows that the limit of galactosamine in total hexosamine can meet the

requirements.

Bacterial endotoxins

Bacterial endotoxins, which originate from the starting material and raw materials, can be

mostly inactivated by oxidation with hydrogen peroxide and removed by filtration.

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The batch analyses show that bacterial endotoxins is controlled within the limit regulated in

the monograph of heparin sodium in current U.S.P. (<0.03 USP Endotoxins Unit/USP

Heparin Unit)

Microorganisms

Microorganisms, which originate from the raw material, can be mostly inactivated by the first

oxidation, the second oxidation and the third oxidation by hydrogen peroxide; it is then

largely removed by 0.22 µm-membrane filtration and 0.1µm-membrane filtration in order. All

the workers, especially for the purification procedure, are dressed and operate strictly

according to the requirement of the clean area. All the equipments and instruments are

controlled to meet the requirements of the cleanliness of the clean area.

Microbiological limit is set for each batch of final product, so each batch of heparin sodium is

to be tested to meet the microbiological requirement before release. Through the above

controls, the final product heparin sodium can meet the specifications in current U.S.P.

Heavy metals

Heavy metals arise from the impurities in the raw materials used in the manufacture.

However, before each batch of materials gains access to the company and before they are

admitted for use, they should be confirmed by the QC of the company that it conforms to its

specifications established strictly. Following a series of steps of purification, heavy metals

can be greatly reduced.

 Possible impurities from the degradation products

The final drug substance is packaged in heat-sealed double-layer medicinal polyethylene bag

and then stored in a sealed aluminum tin. Long-term and accelerated stability study is being

performed. The company promises to complete the accelerated stability study and continue

the long-term stability study, and report any degradation impurity arising from the stability

study through the shelf life.

 Residual solvents

During the manufacturing process of heparin sodium, ethanol is used. According to the

prescriptions of the guideline for Residual Solvents prepared by the International Conference

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on Technical Requirements for Registration of Pharmaceuticals for Human Use (i.e., <467>

Residual solvents, current U.S.P.), ethanol is a Class 3 solvent.

The company tests the residual solvent according to the method of current U.S.P. (see <467>

Identification, control, and quantification of residual solvents), please refer to 3.2.S.4.2 for

the detailed analytical procedure.

The table of impurities profile is shown as follows:

Table of Impurities Profile

Name Structure Source

Protein impurities —— Raw material

Nucleotidic impurities —— Raw material

Dermatan sulfate (DS) Raw material

Oversulfated chondroitin ——
Raw material
sulfate (OSCS)

Galactosamine Raw material

Residual
Ethanol
solvent

 Risk Management Summary (RMS) for Element Impurities

Elemental impurities in drug products may arise from several sources; they may be residual

catalysts that were added intentionally in synthesis or may be present as impurities (e.g.,

through interactions with processing equipment or container/closure systems or by being

present in components of the drug product). Because elemental impurities do not provide any

therapeutic benefit to the patient, their levels in the drug product should be controlled within

acceptable limits.

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According to ICH Q3D guideline on elemental impurities, we use the principles of risk

management described in ICH Q9, conduct risk assessment on the elemental impurities in

heparin sodium of our company, and propose the control strategy to ensure the elemental

impurities are controlled within acceptable limits.

We use the following quality risk management process to conduct risk assessment on the

elemental impurities source and identification, and establish control strategy for elemental

impurities.

Initiate
Quality Risk Management Process

Risk
Assessment Risk Identification

Risk Analysis

Risk Evaluation
Unacceptable

Risk Management cools

Risk Communication

Risk
Control Risk Reduction

Risk Acceptance

Output / Result of the

Quality Risk Management Process

Risk
Review Review Events

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1. Risk Assessment of Elemental Impurities Source and Identification

Flowchart of manufacturing process for Heparin sodium API (injectable grade)

From the above flow chart of heparin sodium manufacturing process, it can be seen that the

denaturation in the manufacturing process is designed to remove most protein and nucleotide

impurities, there is only purification process for crude heparin during the whole process, no

new elemental impurities will arise, so the source of elemental impurities in heparin sodium

API mainly includes the raw material, production equipment , purified water and packaging

materials used in the manufacturing process.

Firstly, the manufacturing process starts from crude heparin, the raw materials used in the

process are shown in the below table.

Table 1 List of Raw Materials

Serial No. Material Name Standard No.

1 Crude heparin N1-STD-R001

2 Recovered ethanol N1-STD-R002

3 Sodium chloride N1-STD-R003

4 Anhydrous sodium carbonate N1-STD-R004

5 Calcium chloride N1-STD-R005

6 Hydrochloric acid N1-STD-R006

7 Sodium hydroxide N1-STD-R007

8 30% hydrogen peroxide N1-STD-R008

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9 Ethanol N1-STD-R009

10 Resin N1-STD-R010

11 Sodium hydrogen sulfite N1-STD-R012

The raw material used in the manufacturing process is crude heparin, the main production

equipment is 316L stainless steel tank, and there are 8 kinds of elemental impurities in the

material, i.e. Mn, Cr, Ni, Cu, Mo, Ti, Fe, V. The raw materials used to manufacturing crude

heparin are sodium hydroxide, sodium chloride, ion-exchange resin, alkaline protease and

ethanol, they are all tested according to the corresponding national standards, after analysis,

there are 5 kinds of potential elemental impurities i.e. As, Heavy metal (Pb), Ca, Mg, Fe.

Therefore, there are totally 12 kinds of potential elemental impurities in crude heparin, i.e.

Mn, Cr, Ni, Cu, Mo, Ti, Fe, V, As, Heavy metal (Pb), Ca, Mg. According to the classification

principle specified in ICH Q3D (see table 2), the potential impurities arising from raw

materials during the production process of heparin sodium API that required to be evaluated

include Cr, Ni, Cu, Mo, V, As, heavy metal(Pb).

Table 2 ICH Q3D elemental impurities classification principles

Whether elements should be

Class Elemental impurities considered in the risk
assessment
Class 1 Cd Pb As Hg Yes
Class 2A Co V Ni Yes
Only when intentionally
Class 2B Tl Au Pd Ir Os Rh Ru Se Ag Pt
added
Depending on the
Class 3 Li Sb Ba Mo Cu Sn Cr
administration route
The raw material used in manufacturing process are all tested as per the current pharmacopeia

specification or industry standards, the potential metallic elements have been tested in

incoming test, the details are shown below:

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Table 3 Materials used in manufacturing process of heparin sodium

and potential elemental impurities

Serial
Material Name Potential elemental impurities
No.
1 Sodium chloride Ba, Ca, Mg, K, Fe, As, Heavy metal
Anhydrous
2 As, Fe, Heavy metal
sodium carbonate
3 Calcium chloride Ba, Al, Fe, Mg, As, Heavy metal

4 Hydrochloric acid Fe, Heavy metal, As

Sodium
5 K, Fe, Al, Heavy metal
hydroxide
30% Hydrogen
6 Ba
peroxide
Sodium hydrogen
7 Fe, Heavy metal, As
sulfite
After analysis, there are totally 8 kinds of potential elemental impurities which may appear in

the raw material, i.e. Ba, Ca, Mg, K, Fe, As, Heavy metal (Pb) and Al. According to the

classification principle specified in ICH Q3D Guideline for Elemental Impurities (see table 2),

the potential impurities arising from raw materials that required to be evaluated includes Ba,

As, Pb.

Secondly, the production equipment which directly contact with the raw materials during the

production process is 316 L stainless steel reaction tank, pipelines and titanium tank, and

there are totally 8 kinds of elemental impurities which exist in the equipment material, i.e.

Mn, Cr, Ni, Cu, Ti, Fe, V. According to the classification principle specified in ICH Q3D

Guideline for Elemental Impurities (see table 2), the potential impurities attributed to

contacting with production equipment and pipelines and required to be evaluated include Cr,

Ni, Cu, Mo, V.

Thirdly, routine testing for purified water which is used in the production process of heparin

sodium is conducted as per pharmacopeia specification, the potential elemental impurities

includes heavy metal (Pb). According to the classification principle specified in ICH Q3D

Guideline for Elemental Impurities (see table 2), the potential impurities arising from purified

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water during the production process of heparin sodium API that required to be evaluated

include Pb.

Lastly, the packing form of heparin sodium API is: firstly sealed in a double-layer medicinal

polyethylene bag, then packaged into aluminum tin, the primary packaging material is a

medicinal LDPE bag, the incoming testing for LDPE bag is conducted according to national

packing material standard, the potential elemental impurities includes heavy metal(Pb).

According to the classification principle specified in ICH Q3D Guideline for Elemental

Impurities (see table 2), the potential impurities arising from packaging materials during the

production process of heparin sodium API that required to be evaluated include Pb.

In conclusion, according to risk assessment of raw materials, production equipment, purified

water and packaging material used in the manufacturing process of heparin sodium, the

elemental impurities include: Ba, Cr, Cu, Mo, As, Pb, V, Ni.

Table 4 Elemental impurities identification for heparin sodium API

Possible sources
that introduce the
Potential elemental impurities in
elemental Potential elemental impurities
heparin sodium and classification
impurities into
heparin sodium
Cr, Ni, Cu, Mo, V, As, Heavy
Crude heparin
metal（Pb）
Auxiliary material Ba, As, Pb
Class 1: As, Pb；
Production
Cr, Ni, Cu, Mo, V Class 2A: V, Ni；
equipment
Class 3: Ba, Cr, Cu, Mo
Purified water Heavy metal（Pb）
Packaging
Heavy metal（Pb）
material
Heparin sodium manufactured by our company is mainly used as the raw material of

injections, according to the Table 5.1: Elements to be Considered in the Risk Assessment

which is specified in ICH Q3D Guideline for Elemental Impurities, the elemental impurities

should be evaluated includes 5 kinds, i.e. Cu, As, Pb, V, Ni, while Ba, Cr and Mo are

unnecessary to evaluate.

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2. Evaluation for Elemental Impurities

According to assessment for raw material, production equipment, purified water and

packaging materials used in the manufacturing process of heparin sodium, there are totally 5

kinds of potential elemental impurities, i.e. Cu, As, Pb, V, Ni. We tested the 5 kinds of

elemental impurities in three batches of heparin sodium, the results are shown below:

Table 5 Testing results for 5 kinds of elemental impurities in heparin sodium API
Acceptable
Batch No. Pb As Cu Ni V Conclusion
criteria
NHS161001 3.349ppb Absent 0.043ppm Absent Absent Pb:≤0.5ppm；
As:≤1.5ppm；
NHS161002 1.314ppb Absent 0.013ppm Absent Absent Cu:≤30ppm； Complies
Ni:≤2.0ppm；
NHS160908 0.852ppb 14.08ppb 0.010ppm 10.04ppb Absent V:≤1.0ppm
According to ICH Q3D Appendix 2: Established PDEs for Elemental Impurities (table A.2.1

Permitted Daily Exposures for Elemental Impurities and table A.2.2 Permitted Concentrations

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of Elemental Impurities for Option), the 5 kinds of elemental impurities in heparin sodium

API are all within the acceptable criteria, and far below 30% of the established PDE in the

drug product.

3. Control Strategy for Elemental Impurities

Through the risk assessment for the potential elemental impurities arising from the

manufacturing process of heparin sodium, we can conclude that the potential impurities are

all within the specified range (see table 5 Testing results for 5 kinds of elemental impurities in

heparin sodium API ), then additional controls are not required for the moment, however, the

incoming testing for raw material and periodical testing for purified water are necessary,

and we will pay close attention to the variation of elemental impurities in product.

4. Risk Management Summary

Through the assessment for the potential elemental impurities arising from the manufacturing

process of heparin sodium, we can conclude that there are totally 5 kinds of potential

impurities, i.e. Cu, As, Pb, V, Ni. Based on the testing result of elemental impurities

obtained by QC, all the above elemental impurities are within the required range, and far

below 30% of the established PDE in the drug product. Additional controls are not required

for the moment, however, the incoming testing for raw material and periodical testing for

purified water are necessary. In addition, we will conduct regular testing of the above 5

kinds of elemental impurities for heparin sodium API and will pay close attention to the

variation of elemental impurities in product. When there is change which has influence on the

content of elemental impurities in product, e.g. changes about raw material supplier, raw

material, equipment, process, etc. re-valuation should be conducted, including the assessment

for the established elemental impurities control method.

Intended route of administration/Use of the substance: parenteral

Element Class Intentionally added Considered in risk management? Conclusion

Cd 1 No Yes Absent

Pb 1 No Yes Absent

As 1 No Yes Absent

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Hg 1 No Yes Absent

Co 2A No Yes Absent

V 2A No Yes Absent

Ni 2A No Yes Absent

Tl 2B No No Absent

Au 2B No No Absent

Pd 2B No No Absent

Ir 2B No No Absent

Os 2B No No Absent

Rh 2B No No Absent

Ru 2B No No Absent

Se 2B No No Absent

Ag 2B No No Absent

Pt 2B No No Absent

Li 3 No Yes Absent

Sb 3 No Yes Absent

Ba 3 No No Absent

Mo 3 No No Absent

Cu 3 No Yes Absent

Sn 3 No No Absent

Cr 3 No No Absent

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