Characterization of PEG Reagents

Before conducting any coupling reaction, it is generally advised that one verify the amount of active moiety in the activated PEG batch. This is important especially when using a reactive PEG from a vial that was opened previously (see Note 2). For this control, several methods are available depending upon the PEG reactive moiety, among which are NMR and colo-rimetric tests.

3.1.1. NMR of PEG Derivatives

Any reactive PEG can be analyzed by both 1H- and 13C-NMR analysis. Both techniques are straightforward and accurate. However, NMR normally requires a high amount of reagent, which is not easily recovered after the analysis. The PEG sample is dissolved in the desired deuterated solvent at 3-5% (w/v) concentration (depending on the molecular weight of the polymer backbone). Particular care needs to be taken in the instrument shim. The best solvents are deuterated dimethylsulfoxide, that is, DMSO-d6, and deuterated chloroform (CDCl3), but others can also be used. As a common procedure, the intensity of the signals of the reactive group are compared with those of the backbone chain (CH2)2 or, better, the terminal methoxy residue (7,16,17).

3.1.2. Colorimetric Assays

Colorimetric assays are used to determine the degree of activation of amine- or thiol-reactive PEGs and PEG-thiols. These assays are as accurate as NMR and often require the consumption of less material. More precisely, the reactivity of PEG-N-hydroxysuccinimidyl (PEG-NHS) esters can be assayed by the "glycil-glycine" test (see Subheading 3.1.2.1.) whereas the amount of free thiols can be determined by direct Ellman's assay (21,22; Subheading 3.1.2.2.). An indirect Ellman's assay can be used to determine the amount of thiol reactive groups (maleimide, vinylsulfone, pyridylthione).

3.1.2.1. Glycil-Glycine Test to Evaluate the Activation of Acylating Groups (NHS and Benzotriazole Reagents)

This test is performed at room temperature.

1. Prepare two vials, mark them 1 and 2, and add, to each of them, 50 |L of 10X gly-gly and 450 |L of borate pH, 8.0.

2. To sample 1, while stirring, add 1 equivalent of PEG-NHS or other acylating group (1 |imol; e.g., if the MW of mPEG-NHS is 5000, add 5 mg and double amount for the 10,000 Da of PEG) in powder form.

3. To sample 2, add nonreactive PEG-OH of any molecular weight in powder form (the same amount in weight as PEG-NHS of sample 1; e.g., if mPEG-NHS in sample 1 is 5 KDa, then add 5 mg of PEG-OH of any molecular weight).

5. Perform the TNBS test according to the method of Snyder and Sobocinsky (see Subheading 3.1.2.1.1., ref. 23).

3.1.2.1.1. TNBS Test According to Snyder and Sobocinsky for NH2 Quantification

Perform the test at room temperature, in duplicate:

1. In separate test tubes, mix 100 |L of sample 1, or sample 2, or borate, pH 8.0 (for the blank); 900 |L of borate, pH 9.3; and 40 |L of TNBS solution 1.

2. Wait 30 min and then, using a spectrophotometer, read the absorbances at 420 nm.

3. Compute: 100 - [(Abs from sample B) - (Abs from blank)]/[(Abs from sample A) - (Abs from blank)] x 100, where Abs is absorbance. This value corresponds to the degree of activation of PEG-NHS in the batch.

3.1.2.2. Direct Ellman's Assay for SH Quantification

This assay (21,22) is used to determine free SH groups in solution and it may be followed to monitor any SH bearing molecule, this being a protein, a polymer, or a low molecular weight compound.

1. Dissolve PEG-SH (or any SH-carrying compound) in oxygen-free phosphate-EDTA, pH 7.0, at a final concentration of 2 mM (if PEG-SH has a MW of 5000, then dissolve it at 10 mg/mL). This solution should be used immediately after preparation.

Perform the test at room temperature, in duplicate:

2. In separate test tubes, mix 30 ||L of PEG-SH solution (or the solution containing SH) or 30 |L of phosphate EDTA, pH 7.0 (as blank), and 970 |L of phosphate-EDTA, pH 7.0.

4. Incubate at room temperature for 15 min, then read Abs at 412 nm.

5. Compute the following: Abs sample - Abs blank. Thiol concentration is calculated knowing that the molar absorbivity (e) of the 5-thio-nitro benzoic acid that forms quantitatively from is SH residue is 14,150 (22).

3.1.2.3. Indirect Ellman's Assay (to Evaluate Thiol-Reactive PEGs)

1. Dissolve the reactive PEG in phosphate-EDTA, pH 6.0, at a final concentration of 2 mM (e.g., if the PEG molecular weight is 5000 Daltons, then dissolve it at 10 mg/mL).

Perform the test at room temperature, in duplicate:

2. In separate test tubes, mix 500 |L of reactive PEG solution or phosphate-EDTA, pH 6.0 (as a blank), and 500 |L of cysteine solution.

4. Perform, on both samples, the direct Ellman's assay as described previously (Subheading 3.1.2.2., step 2).

The amount of thio-reactive mPEG is calculated from the difference in thiol concentration in the two test tubes.

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