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Impd preparation example 2: [68Ga]dota-noc


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IMPD preparation example 2: [68Ga]DOTA-NOC
[68Ga]DOTA-NOC is a well established radiopharmaceutical. Several methods of preparation/purification have been published and adopted by the various involved Small Scale Radiopharmacies. Thus, [68Ga]DOTA-NOC may be prepared using different generators and automated systems, and following different purification protocols; consequently, different reaction/purification conditions may be adopted. The template here described involves the use of a “common” automated module based on pre-assembled kits (“cassettes”), and a pre-purification step via cation exchange cartridges. The proposed example cannot account, for obvious reasons, for all of the above mentioned methods and techniques, and it includes information and data related to one of the possible preparation routes.

It is of paramount importance to remember that, during the preparation of the IMPD, every applicant should include the specific description of their own instrumentation, radiosynthetic pathway, experimental conditions, purification methods, data, etc. and also define their specifications with an appropriate justification.

2.2.1.S DRUG SUBSTANCE
2.2.1.S.1.1 Nomenclature

Drug substance

68Ga-(1,4,7,10-tetraazacyclododecane-N,N ,N ,N-tetraaceticacid)-1-NaI-3-octreotide; 68Ga-DOTA-NOC; 68Ga-DOTA-[Nal3]-octreotide;L-cysteinamide, 68Ga-N-[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]-D-phenylalanyl-L-cysteinyl-3-(1-naphthalenyl)-L-alanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)propyl]-, cyclic (2-7)-disulfide.
Radionuclide: Gallium-68

2.2.1.S.1.2 Structure


Fig. 1: Structure of [68Ga]DOTA-NOC
Molecular formula: 68Ga-DOTA-D-Phe-Cys-1-NaI-D-Trp-Lys-Thr-Cys-Thr(ol), cyclic disulfide

Molecular weight for “cold” reference Ga-DOTA-NOC: 1522.40


2.2.1.S.1.3 General Properties

Physicochemical and biological properties:

[68Ga]Ga-DOTA-NOC or [68Ga]Ga-1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid-(DOTA)-1-NaI3-octreotide is produced by complexing the positron emitting radionuclide radionuclide 68Ga with the chelator DOTA attached to a derivative of the small peptide octreotide. [68Ga]Ga-DOTA-NOC is a somatostatin analog that recognizes somatostatin receptor subtypes 2, 3 and 5 with high affinity. After 68Ga radiolabeling, the receptor affinities remain unchanged or even increased. 68Ga is the daughter nuclide of the so called 68Ge/68Ga generator. Radiophysical properties of 68Ga are summarized in Table 1:




Parent Nuclide

T1/2

Decay mode 

+ Emean

+

γ KeV

Daughter nuclide

68

31


Ga




 67.6 min 

 β+ 

829.5 KeV

88.91%

 511 

 

68
30

Zn

 

Table 1 – 68Ga decay scheme (http://www.nndc.bnl.gov/chart/decaysearchdirect.jsp?nuc=68GA&unc=nds)
The decay scheme of the parent radionuclide 68Ge is depicted in Table 2.


Parent Nuclide

T1/2

Decay mode 

 Emax

Relative

intensity



Daughter nuclide

68

32


Ge




 270.8 days 

 EC 

106 KeV

100%

 

68
31

Ga

 

Table 2 – 68Ge decay scheme (http://www.nndc.bnl.gov/chart/decaysearchdirect.jsp?nuc=68GE&unc=nds) scheme

Table 3 lists representative physicochemical characteristics of the precursor, DOTA-NOC acetate.




Physicochemical characteristics of DOTA-NOC acetate

Appearance

White lyophilized, hygroscopic

Reconstitution

Ultrapure sterile water

pH

After reconstitution with water (pH = 3,5-4,5)

LD50*

20 mg/kg (Rabbit)

Table 3 – List of selected physicochemical properties of DOTA-NOC acetate
*LD50 values are available for DOTA-NOC acetate only
2.2.1.S.2.1 Manufacturer(s)

The preparation of a radiopharmaceutical is usually a continuous process, and the active substance is, as a rule, not isolated. Thus, the information related to the manufacturer will be given in section 2.2.1.P.3.1.



2.2.1.S.2.2 Description of Manufacturing Process and Process Controls

[68Ga]DOTA-NOC is prepared using an automated synthesis module. The generator is eluted with 10 mL of 0.1N HCl, and the eluate is collected to the pre-purification cation exchange column, which is then eluted an with acetone/HCl mixture. The eluate is collected into the reaction vial, previously loaded with DOTA-NOC (50 g in 0.1M acetate buffer solution at pH = 4), where the reaction takes place at 95°C for 5 min. After cooling, 2 mL of physiological saline are added to the reaction vial, and then the mixture is loaded onto a C18 cartridge, which retains [68Ga]-DOTA-NOC and releases free 68Ga, acetone and other possible impurities. After washing the cartridge with physiological saline, [68Ga]DOTA-NOC is then eluted with 1 mL of a mixture of physiological saline/ethanol (1/4). The final product is diluted with physiological saline in order to obtain a final volume of 10 mL. The final product is sterilized by filtration through an inline 0.22 µm filter. Due to the high emission energy of 68Ga and multi-step synthesis, [68Ga]DOTA-NOC is prepared using fully automated radiosynthesis modules. They are capable of performing all the necessary operations, from the elution of generator to the final formulation as an injectable solution of the radiopharmaceutical. For these reasons, these radiopharmaceutical preparations have to be considered as continuous processes carried out in closed systems. As a consequence, the active substance, as well as intermediates or by-products, are as a rule not isolated.


68Ga generator

Specification for the eluate are defined in the European Pharmacopoeia Monograph N°: 2464 (Gallium (68Ga) chloride solution for radiolabelling) and are summarized in Table 4:




68Ga specifications

Appearance

Colorless, clear solution

Radionuclidic purity

≥ 99.9%

68Ge

Less than 0,001%

(10 Bq/MBq of 68Ga)



Non radioactive metal contamination (ICP-EOS)

Iron: <10 µg/GBq

Zinc: < 10 µg/GBq



Identity

Gamma spectrometry: gamma rays at 0,511 ; 1,077 MeV (a peak sum could be observed at 1,022 MeV)

Half-life

62 to 74 min

Radiochemical purity

ITLC-SG plate; mobile phase: methanol/ammonium acetate (1/1) + RP-HPLC results combined: >95%

pH

≤ 2

Endotoxin level

≤ 175 EU/V

Table 4 – Characteristics of 68Ga eluate
2.2.1.S.2.3 Control of Materials

The precursor DOTA-NOC should be manufactured according to GMP for API starting materials. As for the other starting materials, they should be preferably of pharmaceutical grade. In Table 5 a list of the starting materials used in the preparation of [68Ga]DOTA-NOC is presented.



Materials

Test

Cassette (“kit”)

Visual inspection and control of each connection in aseptic condition (laminar flow cabinet class A)

DOTA-NOC

HPLC; identity should conform; See the attached analysis certificate

HCl

See the attached analysis certificate

Sodium acetate

See the attached analysis certificate;

Acetic acid

See the attached analysis certificate

Acetone

See the attached analysis certificate

Water for injections

See the attached analysis certificate

NaCl 0,9% injectable solution

See the attached analysis certificate

Ethanol

See the attached analysis certificate

Table 5 – List of the starting materials used in the preparation of [68Ga]DOTA-NOC
2.2.1.S.2.4 Control of Critical Steps and Intermediates

The “in-process” controls are limited to the monitoring of the critical parameters (e.g. activity, reaction temperatures and pressures) through the graphical control software interface. Printouts of representative preparation process diagrams are usually provided. A full quality control program is set for the final product (see section # 2.2.1.P.5.1.)


2.2.1.S.2.5 Process Validation and/or Evaluation

Please refer to the section 2.2.1.P.3.5


2.2.1.S.2.6. Manufacturing Process Development

Please refer to the section 2.2.1.P.2.3


2.1.2.S.3 Characterization:

2.1.2.S.3.1 Elucidation of Structure and other Characteristics

Non radioactive Ga-DOTA-NOC or DOTA-NOC are used as references. HPLC of reference standards is performed in the same conditions as defined in the European Pharmacopoeia monograph of Ga-DOTA-TOC (Gallium (68Ga) edotreotide injection). No further structure elucidating analyses are required. The experimental conditions are given in the section # 2.2.1.P.5.2


2.1.2.S.3.2 Impurities

Radionuclidic purity:

The same radionuclidic purity required for the 68Ga eluate (see Table 4) is required for [68Ga]DOTA-NOC, the more likely radionuclidic impurity being 68Ge potentially released by the generator.


Radiochemical purity

Analytical method for radiochemical purity determination is analytical HPLC, with integrated radiochemical detector. More details are given in section 2.2.1.P.5.2. The more plausible radiochemical impurity is represented by Ga(III) ions, which may be released by the complex with DOTA-NOC after labeling or as an unreacted species. The other theoretical radiochemical impurities are represented by radiolysis back products. Presence of ethanol during the radiosynthesis and in the final product is aimed to reduce these phenomena.




Chemical purity

The more plausible chemical impurity in [68Ga]DOTA-NOC preparations is acetone, which is used for radiosynthesis (solvent of class C). As for the residual solvents, their limits are defined in the document “EMEA note for guidance on impurities: residual solvents” (CPMP/ICH/283/95), and in the Chapter 5.4 of Ph. Eur. The analysis is usually performed using gas-chromatography. More details are given in the section 2.1.P.5.2.


2.2.1.S.4 Control of the Drug Substance:

Details on methods for [68Ga]DOTA-NOC analysis, their validation, the batch analysis, and the justification of specifications will be provided in the appropriate 2.2.1.P sub-sections.


2.2.1.S.5 Reference Standards or Materials:

The list of the reference standards is provided in Table 6. There are two distinct kind of reference standard: i) chemical standards, ii) radionuclide calibrated sources.

The chemical reference standards are commercially available, chemical grade products. The specifications for purity have been set by the supplier, and accepted by the applicant. Most of the analytical tests (e.g. HPLC, MS) are performed by the supplier, and described in the attached Certificate of Analysis. The chemical reference standards are re-tested for chemical purity by the applicant using HPLC, as detailed in the section 2.2.1.P.5.

The radioactive reference standards are used to verify the calibration status of the gamma spectrometer and the dose calibrator, respectively. Their composition, and the activity(ies) of the radionuclide(s) at reference time and date are described in the attached Certificate of Analysis. The calibrated sources are metrologically referable to recognized standards, and a verification of their identity or purity is in this case non applicable.




Reference standard

Aim

Test

Acceptance criteria

DOTA-NOC

DOTA-NOC Identity

HPLC

Purity> 95%

Multinuclide source

Gamma spectrometer calibration

Not applicabile

Not applicabile

Mononuclide source

Dose calibrator

Not applicabile

Not applicabile

Table 6 - List of reference standards
2.2.1.S.6 Container Closure System

For the reasons already stated above, container closure system will be described for the drug product, in the appropriate sections.


2.2.1.S.7 Stability

For the reasons already stated above, stability will be described for the drug product, in the appropriate sections.



2.2.1.P INVESTIGATIONAL MEDICINAL PRODUCT UNDER TEST
2.2.1.P.1 Description and Composition of the Investigational Medicinal Product:

Each vial contains 10 ± 2 mL of a physiological saline / 8% ethanol solution of [68Ga]DOTA-NOC, with radioactive concentration in the range 20-160 MBq/mL at reference time. Reference time is taken as the time when the activity contained in the finished drug product is measured with the dose calibrator. The [68Ga]DOTA-NOC solution is contained in 15 mL borosilicate glass vials (Ph. Eur. type I), sealed with chlorobutyl rubber stopper (Ph. Eur. compliant), which is in turn secured with an aluminum flip-off cap.


Composition

The composition of the finished drug product is given in Table 7:




Component

Quantity

Function

Reference

[68Ga]DOTA-NOC

20-160 MBq/mL

Active substance

N.A.

NaCl 0,9% solution

9 mL

excipient

Ph. Eur.

Ethanol absolute

0,8 mL

excipient

Ph. Eur.

Water for injection

0,2 mL

excipient

Ph. Eur.

Table 7 – Composition of the injectable solution of 68Ga-DOTA-NOC
2.2.1.P.2 Pharmaceutical Development

[68Ga]DOTA-NOC is one of the possible 68Ga labelled DOTA-peptides, together with [68Ga]DOTA-TOC and [68Ga]DOTA-TATE, and its preparation route may be now considered as well established. Thus, elution of the generator and purification of [68Ga]GaCl3 are performed by following a well known protocol, using 0.1N HCl followed by elution with HCl/acetone which has been demonstrated to be effective in assuring good elution yield and suitable purity of the eluate, with nearly complete removal of potential metal contaminants and 68Ge breakthrough. The preparation of [68Ga]DOTA-NOC has also been set up using an automated synthesis module, by means of which the desired product is purified at the end of the labelling reaction via the well known technique using a C18 cartridge which retains [68Ga]DOTA-NOC and releases free 68Ga and acetone. After washing the cartridge with physiological saline, [68Ga]DOTA-NOC is then eluted with about 1 mL of a mixture of physiological saline solution/ethanol (1/4). The final product is then diluted with physiological saline solution in order to obtain a final volume of 10 mL. The final product is sterilized by filtration through an inline 0.22 µm filter. The active substance is never isolated in the course of the preparation process, and it is available for quality controls only at the end of formulation step.

Stability studies have been performed, demonstrating that the composition of the [68Ga]DOTA-NOC solution does not undergo significant changes to chemical and radiochemical purity during the assigned shelf life of 4 h. For more information about stability, see section 2.2.1.P.8.
2.2.1.P.2.3 Manufacturing Process Development

Not applicable


2.2.1.P.3 Manufacture:

2.2.1.P.3.1 Manufacturer(s)


Institution Name




Address




Person responsible for the small scale preparation of radiopharmaceuticals




e-mail address




Phone




Fax





2.2.1.P.3.2 Batch Formula

A batch of [68Ga]DOTA-NOC is usually represented by a single, multi-dose vial. The materials used in the preparation of a typical batch using the Pharmtracer EZ™ are assembled in a disposable sterile cassette module are listed in Table 8.




Starting materials

Amounts

DOTA-NOC

50 µg

Ultrapure sterile water

0.1 mL

NaCl 0,9% physiological solution

250 mL

Ethanol absolute

1 mL

Ultratrace acetate buffer 0,2 M pH 4

2 mL

Ultratrace acetone/HCl 0,02N mixture (98/2)

2 mL

Ultratrace HCl 0,1 N

6 mL

Specific sterile, single use, cassette module

1

Table 8 – List of starting materials used in a typical [68Ga]-DOTA-NOC batch production
2.2.1.P.3.3 Description of Manufacturing Process and Process Controls

The [68Ga]DOTA-NOC preparation process has already been described in section 2.2.1.S.2.2 of the present document. However, a flow chart is added below:





Fig. 2: Flow chart of the preparation process of [68Ga]DOTA-NOC

2.2.1.P.3.4 Controls of Critical Steps and Intermediates

Description of in-process controls and intermediated has already been given in section 2.2.1.S.2.4


2.2.1.P.3.5 Process Validation and/or Evaluation

Process validation has been performed by preparing and controlling three consecutive batches of [68Ga]DOTA-NOC. Validation runs have been performed in the same operating conditions (e.g. starting 68GaCl3 activity, amount of precursor, physicochemical reaction parameters) characteristic of the process; i.e. using the same instrumentation and starting materials in the stated quantities normally set for typical runs. The parameters evaluated, and their acceptance criteria are reported in Table 9:





Parameters

Acceptance criteria

Radioactive concentration

20 – 160 MBq/mL

Final volume

10  1 mL

Table 9 – Production parameters evaluated during process validation
The experimental data are reported in the following table:





BATCH 1

BATCH 2

BATCH 3

[68Ga]DOTA-NOC activity

1020 MBq

1080 MBq

1007 MBq

Radioactive concentration

102 MBq/mL

108 MBq/mL

101 MBq/mL

Volume

10 mL

10 mL

10 mL

Conform

□ yes□ No

□ yes□ No

□ yes□ No

Table10 – Process validation: experimental data
Each batch has been analyzed following the complete quality control program. The results are summarized in Table 11. The specifications are described in section 2.2.1.P.5.1.



QUALITY CONTROL




Batch 1

Batch 2

Batch 3

Test

Acceptancecriteria

results

Conform

results

Conform

results

Conform

pH

4.0-8.0

5.5

□ Yes □ No

6.0

□ Yes □ No

5.5

□ Yes □ No

Appearance

Clear, colourless

Clear, colourless

□ Yes □ No

Clear, colourless

□ Yes □ No

Clear, colourless

□ Yes □ No

Acetone

≤ 50 mg/V*

< LOD

□ Yes □ No

< LOD

□ Yes □ No

< LOD

□ Yes □ No

EtOH

≤ 400mg/V*

35 mg/mL

□ Yes □ No

34 mg/mL

□ Yes □ No

35 mg/mL

□ Yes □ No

DOTA-NOC

3-5 µg/mL

3,6 µg/mL

□ Yes □ No

3,8 µg/mL

□ Yes □ No

4,2 µg/mL

□ Yes □ No

Radiochemicalpurity

(ITLC-SG and HPLC)

≥ 91% of 68Ga-DOTA-NOC

98%

□ Yes □ No

97%

□ Yes □ No

99%

□ Yes □ No

Radionuclidic purity

99.9%

99.6%

□ Yes □ No

99.9%

□ Yes □ No

99.9%

□ Yes □ No

68Ge

<0.001%

<0.001%

□ Yes □ No

<0.001%

□ Yes □ No

<0.001%

□ Yes □ No

Half-life

62-74min

67min

□ Yes □ No

67min

□ Yes □ No

66min

□ Yes □ No

Filter integrity

Online Pressure test at 2 Bar

conform

□ Yes □ No

conform

□ Yes □ No

conform

□ Yes □ No

Sterility

Ph. Eur. Conform

Sterile

□ Yes □ No

Sterile

□ Yes □ No

Sterile

□ Yes □ No

Bacterial endotoxins

Ph. Eur. Conform (<17,5 EU/mL)

< 5 EU/mL

□ Yes □ No

< 5 EU/mL

□ Yes □ No

< 5 EU/mL

□ Yes □ No

Table 11 – Experimental quality control data for process validation
*= maximum recommended dose (mL)
2.2.1.P.4 Control of Excipients:

2.2.1.P.4.1 Specifications

The excipients used in the preparation process of the finished investigational radiopharmaceutical should meet the specifications reported in Table 12.



Excipient

Specification

0.9% NaCl

Ph. Eur.

Ethanol

Ph. Eur.
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