Dear Researcher,

Hynic-Annexin will be shipped to you frozen along with lyophilized
stannous tricine. Tricine is a modified glycine used in the labeling of
Hynic. The reagents should be stored in a --20^o^C to --80^o^C freezer
(preferentially -80^o^C).

The Hynic-Annexin vials are ready for radiolabeling with Tc-99m (Sodium
Pertechnetate) as follows:

First remove the vials from the freezer at the time of preparation and
allow them to thaw at room temperature (about 5 min). **Note: Please do
not heat the vial**. Second, after thawing please add 30 to 50 mCi (0.5
ml) of Pertechnetate to the annexin vial and gently swirl. Then,
reconstitute a stannous tricine vial with 3.0 mL of saline for injection
followed by immediate removal of 300 uL (about 50 ug), which you will
add to the Pertechnetate/Hynic-Annexin vial. Swirl the vial and incubate
for 15 min. at room temperature, take the sample for quality control,
then the preparation is ready for further dilution. Please note that the
stannous tricine solution expires at the end of the day (12 hr
stability), so, it must be prepared at the time of the labeling.

The radiolabeling yields are greater than 90% in as short of a time as
15 min. Quantification can be performed using ITLC-SG strips developed
in ACD buffer. The amount of activity that stays at the origin of the
plate is counted and compared against the total plate activity.

Attached is additional information, generously provided by Dr. Jean-Luc
Vanderheyden (jlvand@earthlink.net), and concerning the labeling
procedures and answers to some of the typical questions related to the
radiolabeling.

Please contact the BRB-Preclinical Repository if you have any questions.

Good luck with your research,

# Rose Aurigemma 

Rosemarie Aurigemma, Ph.D.

Director, BRB Preclinical Repository

Developmental Therapeutics Program

DCTD, NCI, NIH

Email: raurigemma@ncifcrf.gov

**Additional questions related to Tc-99m Hynic-Annexin radiolabeling
conditions:**

*Q1 - Hynic-annexin V is* *supplied 0.275 mg/vial. I* *cannot use all
the product, since I only need 5 ug/kg* *annexin V for mice imaging, and
I don't have excess Tc-99m.* *Could one* *divide one vial to several*
*aliquots?*

> Yes, you can sub-divide the Hynic-Annexin but you cannot do this for
> the stannous tricine. Here is the recommended protocol:

1.  Thaw a vial of Hynic-Annexin at room temperature. It will only take
    5-8 minutes.

2.  Open the vial by uncrimping the aluminum cover. If you don\'t have a
    decrimper for that size, you can use a pair of scissors, and place
    one blade under the lip of the aluminum foil and the other on top of
    the vial. After a few trials, you should be able to get the aluminum
    only, leaving the stopper sealed on the vial.

3.  Have prepared tubes (the ones in which you will radiolabel),
    preferentially 1.5 mL conical (either screw top or cap), which you
    will have labeled properly with date, concentration and storage
    conditions.

4.  Dispense the amount that you wish to aliquot. We have tested 25 ug
    Hynic-Annexin before. Make sure that your tube is upright, closed
    and place in a -80^o^C freezer immediately. It is important that you
    do these steps quickly in order to avoid decomposition of the Hynic
    and maintain high radiochemical purity.

5.  When you are ready to perform a labeling, take one of these vials,
    and thaw. There is enough tricine in the Hynic-Annexin vial to
    support good radiolabeling yields even if you don\'t use the
    stannous tricine.

*Q2 - About the stannous tricine solution,* *is it possible to aliquot
them since it seems that the reconstituted solution is* *only stable
only for one hour or so?*

> The reconstituted stannous tricine cannot be easily aliquoted. We, and
> our collaborators, have found that it yielded significant amounts of
> TcO2. Note that the purpose of the stannous tricine is to be a source
> of stannous ions for radiolabeling. Therefore, you can make your own
> stock solution of stannous chloride dihydrate (1 mg/mL) in 0.05 M HCl
> and use that instead. Of course, similarly, the stannous chloride
> solution is decomposing with time, but you should have a good supply
> of stannous and diluted HCl. The solution is good for a little longer
> than 1 hr, and has been used for ½ day, but you will see that the tin
> will come out of solution after that period of time.

*Q3 - If* *one Hynic-Annexin* *vial is divided into several aliquots,
vials,* *how does one change* *the condition of labeling?*

> The radiolabeling needs to be adjusted accordingly. You should not
> attempt to do radiolabeling with Tc-99m at greater than 200 uCi/ug.
> Therefore, you will be limited by what you can use in your experiments
> to about 5 mCi Tc-99m. If your elution is \"old\", then it will be
> worse (as it will be quite a bit more of Tc-99g). You should use
> Tc-99m that has been eluted within about 4 hr. Also, don\'t use
> \"first generator eluant\" as there has been impact on stability of
> Tc-99m Hynic-Annexin.

The radiolabeling procedure would then be as follows:

1.  Thaw the Hynic-Annexin aliquot.

2.  Add about 200 uL saline (to give it some volume; this is important
    so that the stannous concentration is diluted)

3.  Add the pertechnetate (we suggest 10 to 30 uL)

4.  Add the stannous solution, about 20ug maximum, either from the
    stannous tricine or from the extemporaneously prepared stannous/HCl.

5.  Mix and cap, wait 15 min.

> Important steps: if you see a precipitate or cloudiness, it is likely
> that the pH is too acid. Therefore, please check the pH at the
> beginning to ensure that it is greater than 5.0. Recommended pH is
> between 5.5 and 6.5. Failure to check the pH may result in the
> denaturation of the protein.

*Q4 -- Regarding quality control, ITLC and ACD buffer:* *is it the acid
citrate dextrose (68mmol/L dextrose, 74mmol/L citrate, pH 5.0)? In*
*certain publications* *saline was used for RCP. Can I use saline?*

> For the ITLC SG, you should activate plates in an 110^o^C oven for 30
> min. as recommended by the manufacturer. It works also with
> \"unactivated\" plates, but the results are not always very tight (+/-
> 2% or so). ACD can be purchased from Sigma/Aldrich (Catalog #C3821),
> or you can make it yourself. It is not very expensive and not much is
> required. Saline has been used in the past, but does not allow for the
> differentiation of hydrolyzed/reduced TcO~2~ that may be formed in the
> product. With ACD, Verbeke et al reported that TcO~2~ would likely
> move on the plate.
>
> Reference: Verbeke K, Kieffer D, Vanderheyden JL, Reutelingsperger C,
> Steinmetz ND, Green AM, Verbruggen A. Optimization of the preparation
> of (99m)Tc-labeled Hynic-derivatized Annexin V for human use. 2003.
> Nucl Med Biol. 30:771-778.
>
> Note: The Radiochemical purity (RCP) should be greater than 90%. It is
> typically 94-95%.

*Q5- How much Pertechnetate activity can be added per vial?*

> The critical factor is the total amount of Tc-99g that is contained in
> the Pertechnetate solution; the older the pertechnetate, the lower its
> specific activity. We recommend keeping the volume of pertechnetate
> low, at 0.5 mL. We recommend you do not use the first generator eluant
> as it may contain oxidants that interfere with the radiolabeling
> process.

*Q6- How long does the Hynic-Annexin vial remain stable?*

> We have about 12-month stability at -20^o^C and over 36 months at
> -80^o^C. The glass transition temperature for the Hynic-Annexin
> product is about -45^o^C, so, any temperature below that will have
> increased stability.

*Q7 - How long does the Tc-99m Hynic-Annexin remain stable?*

> When the Tc-99m Hynic-Annexin is prepared in presence of stannous ions
> and tricine it is stable for about 6 h. To avoid degradation, we
> recommend performing dilution and storage of the product in the
> reaction vial until time of use.

*Q8 - Can the solution be filtered by a 0.22 um filter?*

> It is recommended that you filter the solution through a 0.22 um
> filter and measure the activity loss. For animal studies, a Gelman
> Acrodisk (13 mm) has been used successfully. These are low protein
> binding, and have worked well.

*Q9 - Can the solution be diluted with i.e. NaCl 0.9% and are there
minimum/maximum dilutions?*

> Yes. Once you have done QC testing and you are ready to proceed, you
> can dilute with NaCl 0.9%. There has been a noticeable loss of
> radioactivity by adsorption to the glass vial in cases where the
> solution was diluted below 5 ug/mL. This did not seem to affect the
> purity, but about 25% of the activity was \"hanging\" on the vial.
> Also, it is recommended that you adjust the amount of activity at the
> time of the preparation so that the product is not diluted any further
> than 10-fold, or you may add albumin as part of the diluent.

*Q10 - Do you have a procedure for quality control (RC-purity)?*

> Yes, there are two methods. If only one method is available to you,
> that\'s OK. ITLC is used to determine overall radiochemical yields,
> and HPLC to determine radiochemical purity.

Note that further purification is not needed, nor is it necessary.
However, if you wish to further process the sample, the preparation can
be placed on a conditioned PD-10 (Sephadex G-25) column, and eluted in
PBS. It is better if the PBS is made to contain about 2 mg/mL of tricine
and to use the material quickly after purification.

**For the radiochemical yields by ITLC:**

The following must be prepared and assembled in advance.

1.  Thin layer chromatography developing chamber

2.  Dose calibrator, gamma counter or appropriately calibrated radioTLC
    scanner

3.  Chromatographic Solvent: Place a small amount of ACD buffer \[QC
    Component 1\] in the chamber in order to have solvent at about the
    0.5 cm level above the bottom of the chamber.

4.  Silica Gel Impregnated Glass Fiber Sheets (Gelman): Pre-cut the
    strips to a final dimension of approximately 2 cm wide by 6 to 9 cm
    in height. Activate the plates in an 110^o^C oven for 30 min before
    use; then store in a glass jar with a screw cap. The plates are OK
    for 1-2 day use, so, you need to reactivate for a following day.

> **NOTE: The strips are fragile. use forceps for handling.**

######### Test Procedure 

1.  Carefully remove an ITLC chromatographic strip from a storage
    container using forceps. Using a pencil, carefully mark the origin
    at approximately 1 cm from one end of the strip.

2.  Spot a small drop (2 to 5µl) of product at the origin of the ITLC
    chromatographic strip.

3.  Place the chromatographic strip into the developing chamber. Do not
    immerse the spot into the solvent.

4.  Develop the chromatographic strip, allowing the solvent to ascend to
    about 0.5 cm from the strip top. Remove the strip from the
    developing chamber and allow it to dry.

5.  Place the strip on a clean sheet of paper and either use a
    calibrated radioTLC Scanner or cut the developed chromatographic
    strip into three sections.

An example of radioTLchromatogram is attached below. Integration is
accomplished by totaling the activity of the peak at the origin over
that of the whole strip, and using the formula as described in step 8.

![](media/image1.wmf)

6.  If cutting is chosen, identify the sections as solvent front area
    (a), middle area (b), and origin area (c).

  ----------------------- ----------------------- -----------------------
  **Section name**        **6 cm strip**          **9 cm strip**

  Origin area (c)         0 to 2.75 cm            0 to 4.25 cm

  Middle area (b)         2.75 to 3.25 cm         4.25 to 4.75 cm

  Solvent front area (a)  3.25 to 6 cm            4.75 to 9 cm
  ----------------------- ----------------------- -----------------------

7.  Using the developing system described above, Technetium Tc-99m
    Hynic-rh-Annexin-V remains at the origin and non-protein bound
    Technetium Tc-99m labeled material travel with the solvent front.
    The middle section of the strip is used to verify complete
    separation between product and impurities (less than 5% of the total
    Tc-99m activity should be in this section of the strip). **If the
    middle section is greater than 5%, please repeat the testing in
    duplicates and report all results.**

8.  Using a suitable radioactivity counter (e.g., dose calibrator or
    gamma counter), count each section of the strip. Count long enough
    to determine a statistically significant count or count rate for
    each strip section Calculate the radiochemical purity (percent
    Technetium Tc-99m Hynic-rh-Annexin-V) using the following formula:

> \% radiochemical purity = \[c/(a + b + c)\] x 100

**For the radiochemical purity by HPLC:** use a Superose column (see
typical result below). The UV trace is uninformative, so only the
radioactive trace is provided.

Superdex 75 HR 10/30 column (Pharmacia)

0.05 M Phosphate buffer pH 7.0 containing 0.15 M NaCl and 1 mM sodium
azide

Flow rate: 0.5 mL/min, about 200 psi, column at room temperature

Gamma detection: PE FSA Analyzer

![](media/image2.wmf){width="5.759722222222222in" height="3.45in"}

  ------------- -- -- -------- --------- ----------- ---------- --------- --------- -- --
  Name (Tc-99m)       Start    End       Retention   Height               Area %       
                      (mins)   (mins)    (mins)      (CPM                              

  BP 1                1.10     1.20      1.10        1685.7                            

  BP 2                14.80    14.90     14.80       1528.6                            

  Region 1            16.00    18.40     17.30       4114.3               1.37         

  Region 2            18.50    25.50     20.20       187957.1             92.93        

  BP 3                25.80    25.90     25.80       1671.4                            

  BP 4                26.80    26.90     26.80       2071.4                            

  BP 5                27.80    27.90     27.80       2314.3                            

  BP 6                29.70    29.80     29.70       2142.9                            

  Region 3            31.10    37.40     35.30       6700.0               5.70         

  BP 7                42.00    42.10     42.00       1800.0                            

  3 Peaks                                                                 100.00       

  Total Area                                                                           

  Background                                                    1764.9                 

  Unallocated                                                                          
  Area                                                                                 
  ------------- -- -- -------- --------- ----------- ---------- --------- --------- -- --

*Q11 - I have a large animal to inject, can I combine vials?*

> You can combine vials, just prior to labeling, by transferring
> research material into a larger reaction vial, or, after
> radiolabeling.

*Q12 - What other tests might be important to perform?*

> A visual check of the product is recommended. Also, pH should be
> measured. The pH is typically at pH 6 (between 5 and 7).

***DETERMINATION OF pH***

######### Test Procedure 

1.  Remove a pH strip from its container.

2.  Spot a small drop of product on the three sections of the pH paper.

3.  Within 5 minutes, compare the color of the strip with that of the
    color chart on the box in order to determine the pH of the product.

Record results of the pH measurement.

**If the pH is less than 5 or greater than 7 DO NOT administer the
product.**

***VISUAL ASSAY***

######### Test Procedure 

1.  Swirl the vial in an upright position, and look across the diameter
    of the vial against a black background.

2.  The solution must be clear, colorless immediately after
    reconstitution, and may present slight flocculation over time. Such
    flocculation does not affect the quality of the preparation nor
    preclude its preclinical use since the preparation is filtered prior
    to injection.