Optimization of the output of the single-point modification reaction was carried out by altering one single effect factor and keeping other factors the same in multi-level repeated trials. The effect of each factor on the conformation of reaction products was analyzed using SDS-PAGE and high-performance liquid chromatography (HPLC).

The modification of rLZ-8 with mPEG-SPA (1:12, mol:mol) was carried out in a series of buffer systems at varying pH, namely pH 4.0, 5.0, 6.0, 7.0 or 8.0, and then in pH 8.0 phosphate buffer with varying ionic strengths, at 0.025, 0.05, 0.1 or 0.2 M, at room temperature for 2 h, away from light. The results were analyzed by SDS-PAGE followed by gel imaging after staining with barium iodide and Coomassie Brilliant Blue.

In order to address the effect of time and temperature on the modification reaction, rLZ-8 and mPEG-SPA (1:12, mol:mol) were incubated together in a phosphate buffer system (0.1 M, pH 8.0) at 4, 16, 25 and 37°C, and sampled every 0.5 h for analysis by HPLC, up to 2.5 h.

rLZ-8 and mPEG-SPA in a phosphate buffer system (0.1 M, pH 8.0) at molar ratios of 1:1, 1:3, 1:6, 1:12 and 1:24, were mixed at room temperature for 2 h, away from light. HPLC and SDS-PAGE with barium iodide or Coomassie Brilliant Blue staining were used to monitor the reaction products.

The target product was purified using a Hiload 16/70 Superdex 75 gel chromatographic column with 0.05 M phosphate buffer containing 0.15 M NaCl (pH 7.0) and a flow rate of 1 ml/min. After equilibrating the column with the phosphate buffer for 2 column volumes (120 ml), iso-concentration elution was applied for another 1.5 column volumes, with peak collection at every 2 ml and detection wavelengths of 280, 254 and 215 nm. Every peak was detected using SDS-PAGE and HPLC and the retention times of the confirmed peaks were recorded.

A lymphocyte transformation test (BrdU reagent box) was performed according to the manufacturer's instructions in order to determine the biological activity of rLZ-8 and mPEG-SPA-rLZ-8. The lymphocyte transformation test is based on the fact that lymphocytes stimulated by certain antigens transform and proliferate when re-exposed to the same antigens. The biological activity of antigens is measured by monitoring the proliferation of stimulated cells, and by measuring the incorporation of BrdU into replicating DNA. A chromophore coupled to an anti-BrdU antibody enables spectrophotometric detection of proliferating cells.

We used the Lowry method to measure the protein concentration of the reaction product according to the guidelines of the Chinese Pharmacopoeia 2010 Appendix VIB.

Firstly, the sample (in the form of dry powder) was placed into 50 mM NH₄HCO to prepare a 1 mg/ml sample solution. A 20-μl aliquot of the sample solution was then diluted to obtain a 10-mM solution by adding 100 mM dithiothreitol (DTT) and heating at 56°C for 1 h. After cooling the mixture to room temperature, 250 mM indole-3-acetic acid (IAA) was added to obtain a 25-mM sample solution and this mixture was protected from light for 1 h. After completion of this step, 0.5 μg Trypsin was added and the sample was digested at 37°C for 12 h. Finally, the reaction was ended by adding 1 μl of 10% trifluoroacetic acid (TFA). The digestion products and matrix were mixed at a proportion of 1:3 and dried atmospherically. The peptide mass was detected by PMF analysis in order to identify the PEGylated site on rLZ-8 in the following modes: the mass spectra for the 500–4000 m/z range were determined in the reflection positive ion mode and the 1000–10000 m/z range was determined in the linear positive ion mode.

The results of SDS-PAGE analysis of the PEGylation reaction mixture following barium iodide staining are shown in Fig. 1. As the figure indicates, no modification reaction (indicated by the appearance of higher-MW bands) occurred until pH 5.0, and a weak reaction occurred between pH 5.0 and 7.0. When the pH was >8, the reaction was more efficient and numerous modification products were detected. By contrast, variation of ionic strength hardly affected the modification reaction. Therefore, we chose pH 8.0 as the reaction pH in further studies.

The reaction products were single and relatively unstable at 0.5 h, which indicates the reaction was still in progress (Fig. 2A). Between 1 and 2.5 h, the reaction results appeared to be similar (as shown in Fig. 2B-E) and the reaction products did not change significantly (Fig. 3), indicating that the reaction had reached equilibrium after 1 h. Additionally, the experiment was undertaken at room temperature, since temperature did not have a significant effect on the reaction products (data not shown). As a result, we chose room temperature and 2 h as the reaction conditions.

Fig. 4 illustrates how the molar ratios of rLZ-8 to mPEG-SPA influenced the modification reaction. The modification products became increasingly varied (as indicated by the less discrete appearance of the higher-MW bands) when the molar ratio of rLZ-8 to mPEG-SPA was decreased. Only one type of modification product occurred at the molar ratio of 1:1, so each modification product occupies 100% of the product, with the exception of the remaining unmodified rLZ-8. When the molar ratio was 1:24, there were four modification products and each one contributed to 18.57% of the total mixture (Figs. 4 and 5). For ease of purification, a molar ratio of 1:1 was chosen for single-point modification.

As shown in Fig. 6A and Fig. 7, only one type of modification product occurred at a molar ratio of 1:1. According to the molecular sieve principle, the purity of the purification product may reach 99% after being purified using a Superdex 75 gel chromatographic column, a result that we also observed when using this chromatographic purification method (Fig. 6B).

The biological activities of rLZ-8 and PEG-rLZ-8 (as assessed using a BrdU incorporation assay) are shown in Fig. 8. When modified by PEG, the activity of the modification products of rLZ-8 decreased as expected.

The Lowry method was used to determine the concentration of protein in the modification product due to its outstanding linear correlation, both before and after modification. The amount of rLZ-8 decreased from 5 to 0.96 mg following the modification and purification process.

Several points were considered in order to correctly identify the modification site. Firstly, the PEG-peptide may not match the expected peptide mass fingerprint (PMF) and those that match usually represent unPEGylated peptides in the modification sample. Secondly, the PEGylated site is always located at the N-terminus or on a Lys side chain. Thirdly, PEGylated Lys is usually intractable to enzymatic cleavage, in which case, when Lys is modified at least one uncleaved site is always included, and the margin between the PEG-peptide and PEG should be close to the theoretical MW of the peptide, while the MS/MS peak shape of the PEG-peptide should be consistent with that of PEG.

From the data, we conclude that Lys is not the PEGylated site, as all Lys in the PEG-protein matched the PMF and the molecular weight of the PEG-peptides were close to that of PEG. Therefore, we concluded that the PEGylated site did not fall within the 75–111 amino acid fragment of rLZ-8 (this fragment is 4205.9858 Da). In the determination of the PMF of the PEG-protein, N-terminal peptides with and without the M fragment (SDTALIFR) were observed, so the PEGylated site is estimated to be at the N-terminus, as the falling off of M in the process is not expected to affect the correspondence in MW between the peptides and the PMF (Fig. 9). Furthermore, the margin of the MW between PEG-peptides and PEG are relatively close to M, which provides further evidence that the PEGylated site is located at the N-terminus.