IMPD preparation example 3: [177Lu]DOTA-peptide

The present template has been drafted with the aim to provide a suitable track in
case of preparation of a useful therapeutic radiopharmaceutical. 177Lu has been
selected, among other suitable therapeutic radionuclides, due to its particularly
favourable and well known properties, but it has to be clear that other radionuclides
might be selected as well. As for the ligand, a generic peptide has been chosen,
instead of a specific molecule, due to the high number of possible
radionuclide/chelator/ligand complexes which have, on the other hand, a common
preparation pathway. A generic 177Lu labelled DOTA-peptide was selected since
DOTA is one of the most popular and commonly used chelators with high affinity for
the radionuclide. As a consequence, most of the following sections do not contain
real, experimental data, as shown with the other two templates dedicated to
[11C]choline and [68Ga]DOTA-NOC. However, it is the opinion of the authors of the
present document that the information included here is a useful guide to the
preparation of an IMPD for similar radiopharmaceuticals. Indeed [177Lu]DOTApeptides, as well as other suitable therapeutic radiopharmaceuticals, may be
prepared using different radionuclide sources, using automated system or even
manually, and the automated system may be based on cassettes or not. The
template presented here has been prepared hypothesizing the use of a common
automated module. The proposed example cannot account, for obvious reasons, for
all of the above mentioned methods and techniques, and it includes information
related to one of the possible radionuclide/chelator/peptide preparation routes.

It is of paramount importance to remember that, in 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
177
Lu-(1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid)-peptide; [177Lu]LuDOTA-peptide
Radionuclide: Lutetium-177
2.2.1.S.1.2 Structure

Fig. 1 Structure of [177Lu]DOTA-peptide

Molecular formula: CxHy177LuNwOzS
Molecular weight of peptide: _____

2.2.1.S.1.3 General Properties

Physico-chemical properties:
Description of [177Lu]DOTA-peptide (any relevant information should be briefly stated,
e.g. receptors affinity, therapeutic indications, etc.).
Nuclear properties of 177Lu are summarized as follows (Table 1):
Half-life:
6.734 days
Principal Photons:
0.208 (11%), 0.113 (6.4%) MeV
Maximum Beta Energy:
0.497 MeV (79%)
Maximum Range of Beta in Air:
1.35 m
Maximum Range of Beta in Water:
1.6 mm
Half-Value Layer for Lead:
0.6 mm
Table 1 Physical properties of 177Lu (http://ie.lbl.gov/toi/nuclide.asp?
iZA=710177)
Tables 2 and 3 list representative physicochemical characteristics of the precursor
DOTA-peptide and the radionuclide.
Physicochemical characteristics of DOTA-peptide
Appearance
Related information of DOTA-peptide
Reconstitution
Related information of DOTA-peptide
pH
Related information of DOTA-peptide
LD50
Related information of DOTA-peptide
Table 2 List of selected physicochemical properties
177

Lu specifications (obtained from the producer)

Appearance
Colorless, clear solution
177m
Lu (HPGe)
Less than 0,05%
as of production date
All other impurities (HPGe)
Less than 0,01%
as of production date
Non-radioactive metal
Iron: < 20 g/Ci
contamination (ICP)
Zinc: < 40 g/Ci
Aluminum: < 30 g/Ci
Copper: < 30 g/Ci
Calcium: < 100 g/Ci
Lead: < 20 g/Ci
Radionuclidic purity (HPGe)
99%
pH
1-3
Endotoxine level
<5 EU/mL
Table 3 Characteristics of 177Lu
2.2.1.S.2.1 Manufacturer(s)
(To be filled in with data of DOTA-peptide supplier/manufacturer)
Institution name
Address
Responsible person
e-mail address
Phone
Fax

(To be filled in with data of 177Lu supplier/manufacturer)

Institution name
Address
Responsible person
e-mail address
Phone
Fax
(To be filled in with data of [177Lu]DOTA-peptide producer small scale preparation
institution)
Institution name
Address
Person responsible for
the small scale
preparation of
radiopharmaceuticals
e-mail address
Phone
Fax
Table 4 Details of manufacturer(s)
2.2.1.S.2.2 Description of Manufacturing Process and Process Controls
A description from the supplier/manufacturer of the precursor should be added.
A description from the supplier/manufacturer of the 177Lu should be added.
[177Lu]DOTA-peptide is prepared from precursor DOTA-peptide and 177Lu chloride,
which is withdrawn from activity vial and transferred to the reaction vial with
corresponding amounts of peptide and labelling buffer. After the incubation at
selected temperature, the desired product is purified at the end of the labelling
reaction using a C18 cartridge which retains [ 177Lu]DOTA-peptide and releases free
177
Lu. After washing the cartridge with physiological saline, the [ 177Lu]DOTA-peptide is
eluted with a mixture of water/ethanol. The final product is then diluted with saline.
The final product is sterilized by filtration through an inline 0.22 m microbiological
filter.
Due to the high emission energy of 177Lu and multi-step synthesis, [177Lu]DOTA-peptide is prepared using fully automated radiosynthesis modules. They are
capable to perform all the necessary operations, from withdrawing 177Lu from the
activity vial to the final formulation as an injectable solution of the
radiopharmaceutical. For these reasons, the 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.
2.2.1.S.2.3 Control of Materials
A description regarding Control of Materials from the supplier/manufacturer of the
precursor should be added.
A description regarding Control of Materials from the supplier/manufacturer of the
177
Lu should be added.

The precursor DOTA-peptide 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 [177Lu]DOTA-peptide is reported.
Material
Cassette (kit)

Test
Visual inspection and control of each connection in
aseptic condition (laminar flow cabinet class A)
See the attached analysis certificate.
See the attached analysis certificate.
See the attached analysis certificate.

DOTA-peptide
HCl
Ingredients for
corresponding buffer
Water for injections
See the attached analysis certificate.
NaCl 0,9% injectable
See the attached analysis certificate
solution
Ethanol
See the attached analysis certificate.
Any others
See the attached analysis certificate.
Table 5 List of the starting materials used in the preparation of [177Lu]DOTA-peptide

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.4.2.)
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
A description regarding Manufacturing Process Development
supplier/manufacturer of the precursor should be added.

from

the

A description regarding Manufacturing Process

supplier/manufacturer of the 177Lu should be added.

from

the

Development

For [177Lu]DOTA-peptide 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
A description regarding Characterization from the supplier/manufacturer of the
precursor should be added.
Non radioactive Lu-DOTA-peptide and unchelated DOTA-peptide are used as
references and are provided with a suitable Certificate of Analysis. HPLC analysis of
reference standards is performed using the same conditions as defined in the
suitable European Pharmacopoeia monograph or the producer specifications in case
a monograph does not exist. 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:
A description regarding Impurities from the supplier/manufacturer of the
should be added.

177

Lu

Radiochemical purity
The analytical method for radiochemical purity determination is HPLC with integrated
radioactivity detector. More details are given in section 2.2.1.P.5.2. The most
plausible radiochemical impurity is Lu(III) ions as an unreacted species. The other
potential radiochemical impurities are represented by radiolysis products.
Chemical purity
The most plausible chemical impurity in [ 177Lu]DOTA-peptide preparations is ethanol,
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
Chapter 5.4 of Ph. Eur. The analysis is usually performed using gas-chromatography.
More details are given in the section 2.2.1.P.5.2.
2.2.1.S.4 Control of the Drug Substance:
A description regarding Control of the Drug
supplier/manufacturer of the precursor should be added.

Substance

from

the

Details on methods for [177Lu]DOTA-peptide analysis, their validation, the batch

analysis, and the justification of specifications will be provided in the appropriate
2.1.P sub-sections.
2.2.1.S.5 Reference Standards or Materials:
A description regarding Reference Standards
supplier/manufacturer of the 177Lu should be added.

or

Materials

from

the

The list of the reference standard is provided in Table 6. There are two distinct kind of
reference standard: i) chemical standard, 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. The specifications and 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 not applicable.
Reference standard
DOTA-PEPTIDE
Mononuclide source

Aim

Test

DOTA-PEPTIDE
HPLC
Identity
Dose calibrator
Not applicable
Table 6 - List of reference standards

Acceptance
criteria
Purity> 95%
Not applicabile

2.2.1.S.6 Container Closure System

For the reasons stated above, container closure system for the [177Lu]DOTA-peptide
will be described for the drug product, in the appropriate sections.
2.2.1.S.7 Stability
A description regarding Stability from the supplier/manufacturer of the DOTApeptide should be added.
A description regarding Stability from the supplier/manufacturer of the
be added.

177

Lu should

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 X mL of a mixture physiological saline/ethanol (9/1) solution of
[177Lu]DOTA-peptide, with radioactive concentration in the range X - Y MBq/mL at
reference time. The [177Lu]DOTA-peptide solution is contained in X 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
[ Lu]DOTA-peptide X - Y MBq/mL Active substance N.A.
NaCl 0,9% solution
X mL
Excipient
Ph. Eur.
Ethanol absolute
X mL
Excipient
Ph. Eur.
Water for injection
X mL
Excipient
Ph. Eur.
177
Table 7 Composition of the injectable solution of [ Lu]DOTA-peptide
177

2.2.1.P.2 Pharmaceutical Development

[177Lu]DOTA-peptide is prepared using an automated synthesis module, where 177Lu
chloride is withdrawn from the activity vial and transferred to the reaction vial with the
corresponding amount of peptide and labelling buffer. After the incubation at
appropriate temperature the desired product is purified using a C18 cartridge which
retains [177Lu]DOTA-peptide and releases free 177Lu. After washing the cartridge with
physiological saline, [177Lu]DOTA-peptide is eluted with a mixture of water/ethanol.
The final product is then diluted with physiological saline to a final volume of X
mL and sterilized by filtration through an inline 0,22 m microbiological filter. The
active substance is not isolated in the course of the preparation process, and it is
available for quality control only at the end of the formulation step.
Stability studies have been performed, demonstrating that the composition of
the [177Lu]DOTA-peptide solution does not undergo significant changes to chemical
and radiochemical purity during the assigned shelf life (e.g. 24 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 [177Lu]DOTA-peptide usually consists of a single, multi-dose vial. The
materials used in the preparation of a typical batch using automated module are
listed in Table 8.
Starting materials
Amounts
DOTA-peptide
X g
Sterile water (appropriate quality)
X mL
NaCl 0,9% physiological solution
X mL
Ethanol absolute
X mL
Labelling buffer
X mL
Specific sterile, single use, cassette 1
Table 8 List of starting materials used in a typical
[177Lu]DOTA-peptide batch production
Batches of [177Lu]-DOTA-peptide to be used in clinical trials typically consist of a
single vial.
2.2.1.P.3.3 Description of Manufacturing Process and Process Controls
The 177Lu-DOTA-peptide 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 Flowchart of the preparation process of [177Lu]DOTA-peptide

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 testing three consecutive
batches of [177Lu]DOTA-peptide. Validation runs have been performed in the same
operating conditions (e.g. starting 177LuCl3 activity, amount of precursor, reaction
parameters) 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
X Y MBq/mL
Final volume
X Y mL
Table 9 Production parameters evaluated during the process validation
The experimental data are reported in the following table:

177

[ Lu]DOTA-peptide activity

Radioactive
concentration
Volume
Conform

BATCH 1
___ MBq

BATCH 2
___ MBq

BATCH 3
___ MBq

___ MBq/mL ___ MBq/mL ___ MBq/mL

___ mL
yes no

___ mL
yes no

___mL
yes no

Table 10 Experimental production data for process validation

Each batch has been analyzed following the complete quality control program. The
results are summarized in Table 11. Specifications are described in section
2.2.1.P.5.1.

QUALITY CONTROL
Batch 1

Batch 2

Batch 3

Test

Acceptance
criteria

results

Conform

results

Conform

results

pH

4.5-8.5

___

Yes No

___

Yes No

___

Yes
No

Appearance

Description of
solution (i.e.
clear solution)

___

Yes No

___

Yes No

___

Yes
No

EtOH

400 mg/V*

___

Yes No

___

Yes No

___

DOTA-peptide

X - Y g/mL

___

Yes No

___

Yes No

___

X% of
[177Lu]DOTA-

___

Yes No

___

Yes No

___

Radiochemical
purity
Radionuclidic
purity

Half-life

Filter integrity
Sterility
Bacterial
endotoxins

Conform

Yes
No
Yes
No
Yes
No

peptide
99%

6.65 days
Online
Pressure test
at 2 Bar
Ph. Eur.
Conform
Ph. Eur.
Conform
(<17,5
EU/mL)

See the
attached
analysis
certificate
See the
attached
analysis
certificate

Yes No

Yes No

See the
attached
analysis
certificate
See the
attached
analysis
certificate

Yes No

Yes No

See the
attached
analysis
certificate
See the
attached
analysis
certificate

Yes
No

Yes
No

___

Yes No

___

Yes No

___

Yes
No

___

Yes No

___

Yes No

___

Yes
No

___

Yes No

___

Yes No

___

Yes
No

Table 11 Experimental quality control data for process validation

*V = 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.
Table 12 List of excipients
The 0.9% NaCl and ethanol solutions are approved following a check for packaging
integrity, expiry and Certificate of Analysis. The appearance should also be verified.
For the above reasons, for excipient control there is no need to describe analytical
procedures, as well as their validation and justification of specification(s).

2.2.1.P.5 Control of the Investigational Medicinal Product:

10

2.2.1.P.5.1 Specifications
Each batch of [177Lu]DOTA-peptide is submitted to quality control, with the aim to
evaluate chemical, radiochemical and microbiological purity of the finished product.
The QC tests are summarized in Table 13.
Parameters
Appearance

Test
Visual
inspection

Identification

HPLC

Radiochemical purity
Residual solvent*

HPLC
GC

Radionuclidic purity

Half-life
pH
Bacterial endotoxins*
Filter integrity
Sterility*

See the
attached
analysis
certificate
See the
attached
analysis
certificate
pH paper

Specification
Description of solution (i.e. clear solution).
The
principal
peak
in
the
radiochromatogram obtained with the test
solution of 177Lu-DOTA-peptide has
approximately the same retention time as
the principal peak in the chromatogram
obtained with a reference solution of cold
Lu-DOTA-peptide.
[177Lu]DOTA-peptide X%
Ethanol 400 mg/max. injectable volume
177

Lu 99%

6.65 days
X-Y

Ph. Eur.

<175 EU/max. injectable volume

Bubble point

Following specification of the supplier

Ph. Eur.

Sterile

Table 13 Specifications and acceptance criteria for [177Lu]DOTA-peptide

*Tests performed after the release of the radiopharmaceutical, due to both the test
duration not compatible with radionuclide half-life (e.g. sterility test), and for radiation
protection reasons (e.g. bacterial endotoxins test).
2.2.1.P.5.2 Analytical Procedures
Analytical procedures are described in details in relevant Standard Operating
Procedures, which are available on request.
2.2.1.P.5.2.1 Identification and determination of the chemical and radiochemical
purity using HPLC
Instrumentation:
HPLC (description of the instrument, detectors)
[177Lu]DOTA-peptide analysis: specifications
Identification: the main radiochemical peak should
have the same retention time evidenced by the SST test for standard LuDOTA-peptide 0.2 min
Radiochemical purity: peak area for [177Lu]DOTApeptide has to be > X % of the total peak areas
Chemical purity (DOTA-peptide): the peak area for
DOTA-peptide should not be more than the corresponding area of the
peak obtained with reference SST solution

11

2.2.1.P.5.2.2 Determination of residual solvents using gas-chromatography

Instrumentation:
Gas-chromatograph (description of the instrument, operative conditions)
[177Lu]DOTA-peptide analysis: specification
-

Ethanol: maximum admitted concentration = 400 mg/V, where V is the

maximum recommended dose (mL).

2.2.1.P.5.2.3 Determination of pH
Instrumentation: pH paper
- Put 50 L of [177Lu]DOTA-peptide on the pH paper strip and compare the
obtained color with the reference.
[177Lu]DOTA-peptide analysis: specification
pH of the test solution should be in the range X - Y
2.2.1.P.5.2.4 Endotoxins
Instrumentation:
Endosafe PTS Reader
[177Lu]DOTA-peptide analysis: specifications
Bacterial endotoxin level should be < 175 EU/V, where V is the maximum
recommended dose (mL).
2.2.1.P.5.2.5 Filter Integrity Test
This test is required if the finished product is sterilized using 0.22 m filter
membranes.
Instrumentation:
- Specific program is design on the synthesis module in order to limit the
irradiation of the operators. As for the test, a 2 bar pressure is applied
upstream to the filter, and the pressure drop is measured for 1 min.
Method
- Connect a suitable gas source to the synthesis module
- Verify that the pressure is at 2 bar
- Record the pressure values
[177

Lu]DOTA-peptide: specifications
In general the record must conform with the following graph; in particular pressure
should be > 1.0 bar between steps 4 to 5 and 7 to 9 holding pressure for predefined
time after pressure supply is closed.

Fig. 3: Reference pressure vs time graph for the bubble point test

12

2.2.1.P.5.3 Validation of Analytical Procedures

The reference guidelines are as follows:

ICH Harmonised Tripartite Guideline Text on validation of analytical

Procedures Step 4 of the ICH Process, November 2005

ICH Harmonised Tripartite Guideline Validation of Analitycal Procedures:

Text and Methodology Step 4 of the ICH Process, November 2005

The above guidelines may not always apply to validation of radioactive compounds,
due to their peculiar nature. Exceptions will be discussed.
2.2.1.P.5.3.1 Method validation for the determination of chemical purity using
HPLC
Validation of analytical method for the determination of chemical purity of injectable
solution of [177Lu]DOTA-peptide is here presented.
In Table 14 the validation parameters and their acceptance criteria are summarized:
Acceptance criteria

Test
Specificity

2,5
CV % 2%

Repeatability
Intermediate precision

Fcalc value of Ftab

R2 0,99

Linearity
Quantification Limit (LOQ)

CV % 2% for standard
DOTA-peptide
CV % 5% for impurity

Limit of detection (LOD)

---

Robustness

Fcalc value of Ftab

Table 14 Test and acceptance criteria for the validation of the method for the
determination of chemical purity using HPLC
Specificity
Specificity determination is performed analyzing mixture containing critical
components that might be present in the finished product [ 177Lu]DOTA-peptide
solution, and demonstrating that the method can distinguish the various components
present at the limit concentration for the considered standards. The [ 177Lu]DOTApeptide preparation method development did not prompt for chemical impurities,
except for the free 177Lu. Thus, analyses were performed using a series of solution
containing 177Lu-DOTA-peptide, and 177Lu-DTPA.
Peak resolution may be calculated using the following equation:

Rs

1,18 Trb Tra

Wa Wb

where:
Trb= retention time of the compound b
Tra= retention time of the compound a

13

Wb= full width at half-height of the compound b

Wa= full width at half-height of the compound a
Linearity
The statistical function used in these cases is linear regression with least squares.
The curve equation, the correlation coefficient and the determination coefficient (r 2)
are then calculated.
Equation:
y = ax + b
where:
a = slope
b = intercept
y = peak area
x = analyte concentration
r2 should be 0,99 within the concentration operating range.
The above evaluation requires the preparation of at least 5 standard solutions with
different concentrations for each of the analytes of interest (Lu-DOTA-peptide and
DOTA-peptide). Such solutions are usually prepared by serial dilution starting from a
mother solution, with the highest concentration.
Precision
Precision may be considered at different levels, as a measure of repeatability or
intermediate precision.
a. Repeatability:
Repeatability may be calculated based on the content of standard Lu-DOTA-peptide
and DOTA-peptide. The statistical parameter of concern is the variation coefficient
(CV%) (or Relative Standard Deviation. RSD), which is determined using the
following equation:

CV %

s
100
m

where:
s = standard deviation of the peak areas
m = average of the peak areas
CV% should be < 2% for the standard Lu-DTPA, and < 5% for the [177Lu]DOTApeptide and DOTA-peptide.
The necessary experimental data may be obtained by injecting 5 times a
sample of the desired analyte whose concentration should fall within the range
established during linearity test.
b. Intermediate precision
Intermediate precision may be determined through the variance calculation (ANOVA),
which allows, in turn, for Fisher value calculation.
Limit of quantitation (LOQ)
Experimentally, LOQ may be determined by analyzing a series of diluted solutions of
Lu-DOTA-peptide and standard DOTA-peptide, until a concentration level quantified
with a precision > 95% is reached.
The experimental value determined as above described need to be confirmed
through a precision analysis, using a sample at the concentration corresponding to
the found LOQ. Acceptance criterion is CV% < 5%.
Limit of detection (LOD)
The LOD may be determined experimentally by successive dilutions, until the above
minimum concentration is found.

14

Robustness
In the case of HPLC analysis, a critical parameter might be the mobile phase flow.
Once the critical parameter has been selected, three consecutive analyses of the
desired analyte have to be performed, following a deliberate modification of the e.g.
pump flow.
Data may be evaluated through the variance analysis (ANOVA). In the
present document, the parameters whose variation will be analyzed are retention
time and peak areas.
2.2.1.P.5.3.2 Method validation for the determination of radiochemical purity
using HPLC
Validation of the analytical method for the determination of the radiochemical purity is
here presented. In Table 14, the validation parameters and their acceptance criteria
are summarized:

Test
Specificity
Repeatability
Intermediate precision
Linearity

Acceptance criteria
Not applicable
CV % 2%
Not applicable
R2 0,99

Limit of quantitation (LOQ)

Not applicable

Limit of detection (LOD)

Not applicable

Robustness

Not applicable

Table 15 Test and acceptance criteria for the validation of the method for the
determination of radiochemical purity using HPLC

In case of validation of methods of radioactive compounds, some of the ICH

guidelines validation parameters may not be of concern and do not apply. In
particular:
-

Specificity would require the analysis of at least two radioactive analytes with
comparable activity, and this is usually not applicable in case of pure
radiolabelled compounds such as [177Lu]DOTA-peptides.

Flow cell radiochemical detectors typically used in the analysis of

radiolabelled compounds are inherently not very suitable for quantitation
purposes; moreover, they are typically used to determine relative ratios
between the activity of the various labelled compounds that might be present
in the sample, rather than for true activity quantitation purposes, and the final
required outcome is a percentage areas calculation. Thus, both LOQ and
LOD analyses have been considered of no concern.

Similar considerations may be done for intermediate precision analysis, which

is strictly related to the possibility of quantitation of the desired analytes.

As for robustness, the results obtained during the tests for validation of
chemical purity using cold analytes (Lu-DOTA-peptide and standard DOTA-

15

peptide) provide response and data that may apply to the radioactive analytes
as well. A repetition of the test would in this case represent a useless
radiation burden for the operators.
-

Last but not least, the ALARA (as low as reasonably achievable) radiation
protection concept should indeed always be kept in mind while designing the
necessary validation tests.

Linearity
As the half-life of 177Lu is significantly longer, compared with that of radionuclides
typically used in the preparation of diagnostic RPs, the typical experimental approach
based on the preparation of a series of solutions with different concentrations
applies. Thus, 5 solutions with different concentrations have been prepared and
analyzed. However, activity measurements have been corrected for decay. r2 may
thus be extrapolated from the calibration curve by analyzing 5 different radioactive
concentrations of [177Lu]DOTA-peptide.
Repeatability
In the case of repeatability, despite of the relatively long half-life of 177Lu, the effect of
decay may nonetheless be significant. For this reason, repeatability may be
evaluated analyzing a series of HPLC runs obtained with repetitive injections of a
single [177Lu]DOTA-peptide sample, and recalculating the obtained peak area values
with the decay equation:
lnA0= lnA + xt
= 0,693/t1/2
where:
A0= corrected peak area
A= measured peak area
t= time interval between the considered injection and the first one
t1/2= half-life (Lu-177= 6.65 days)
The peak area values normalized for decay, may then be compared and yield a
consistent statistical analysis. Average, standard deviation and (CV%) are then
calculated. Repeatability has to be determined on three different days, to verify the
instrument outcome during the time course.
2.2.1.P.5.3.3 Validation of the analytical method for the determination of
residual solvent using gas-chromatography
Gas-chromatography is used to evaluate the amount of residual solvents in the
finished product solution of 177Lu-DOTA-peptide.
The validation parameters are practically the same as already described for the
validation of the method for the determination of chemical purity (see Table 14,
section 2.2.1.P.5.3.1). For this reason, discussion will not be repeated in this context.
2.2.1.P.5.4 Batch Analyses
Data related to the [177Lu]DOTA-peptide batches are included in the respective
Certificates of Analysis. They report information related to the number and batch size,
as well as information on the production site, methods of production, quality control
and acceptance criteria.
2.2.1.P.5.5 Characterization of Impurities

16

The discussion about the possible impurities in the injectable solution of the finished
product [177Lu]DOTA-peptide has already been described in section 2.2.1.S.3.2,
related to the active substance.
2.2.1.P.5.6 Justification of Specification(s)
The analytical test, the methods and acceptance criteria have been derived, where
applicable, from the general Ph. Eur. monograph Radiopharmaceuticals.
Depending on the specific DOTA-peptide complex used, add information from the
literature in order to justify peptide amount, radiochemical purity and other relevant
parameters.
2.2.1.P.6 Reference Standards or Materials
Reference standards and materials have already been discussed in section
2.2.1.S.5.
2.2.1.P.7 Container Closure System
The radiopharmaceutical product is contained in a glass vial, Ph. Eur. type I, sterile
and pyrogen free, covered with a bromo (or chloro) butyl rubber stopper, sealed with
a flip-off aluminum cap. Attached are the Certificates of Analysis of the vials and
stopper manufacturer. No further tests other than visual inspection are applied to
these materials.
2.2.1.P.8 Stability
Stability studies have been performed to provide evidence on how the quality of
[177Lu]DOTA-peptide may vary with time and to establish a shelf life for the finished
product under recommended storage conditions. During the entire shelf-life, the
radiopharmaceutical characteristics of purity have to meet quality criteria discussed
and established in the previous sections.
The goal is thus to define expiry time and date for the radiopharmaceutical
product. For stability study purposes, three consecutive batches of [177Lu]DOTApeptide have to be prepared by using the maximum possible starting activity of the
radionuclide 177Lu, in order to obtain batches with the highest radioactive
concentration, that allow to evaluate the effect of radiolysis of the active substance in
worst case conditions.
For each batch of radiopharmaceuticals, the following information has to be provided:
- batch number
- preparation date and time
- calibration time
- activity at calibration time
- radioactive concentration at calibration time
Specifications and acceptance criteria for each batch are a part of those defined in
section 2.2.1.P.5.1, table 13. The test program follows the matrix listed below:

17

T0

T+ 4h

T
+24h

Parameters

Test

Specification

Appearance

Visual
inspection

Identification

HPLC

Description of the solution (i.e.

clear, colourless solution)
The principal peak in the
radiochromatogram obtained
with the test solution of 177LuDOTA-peptide has
approximately the same
retention time as the principal
peak in the chromatogram
obtained with a reference
solution of cold LutetiumDOTA-peptide.

Radiochemica
l purity

HPLC

pH

pH paper

X-Y

Bacterial
endotoxins*

Ph. Eur.

<175 EU/ max. recommended

dose (mL)

Filter integrity

Bubble point
test

Following specification of the

supplier

Sterility*

Ph. Eur.

Sterile

177

Lu-DOTA-peptide X%

Table 16 Matrix of the test to be performed during stability study (X means: to be

tested; / means: not to be tested)

18