Stability of trastuzumab during nanomedicine formulation using SEC-HPLC coupled with polyacrylamide gel electrophoresis

Figures & data

Figure 1. Schematic structure of trastuzumab (MW 145 kDa) and its fragments. F(ab)’, antigen binding fragment (MW ∼50 kDa); F(ab)₂’, two F(ab)’ linked by disulphide bonds (MW ∼110 kDa); Fc: crystallisable fragment; C: constant domain; V: variable domain; H: heavy chains; L: light chains.

Figure 2. UV-vis scan and typical SEC-HPLC chromatograms of trastuzumab and F(ab)’. (A) UV scan of trastuzumab (100 µg/ml) and F(ab)’ (generated from 10 mg/ml trastuzumab) peaks with a DAD on SEC-HPLC, indicating suitable detection of both compounds at the wavelength of 278 nm. (B) SEC-HPLC chromatograms of 100 µg/ml trastuzumab showing a pure peak at 6.3 ± 0.1 min, and its F(ab)’ fragment (confirmed by SDS-PAGE) at 7.7 ± 0.1 min. Solvent peaks appeared after 9 min and did not interfere the detection of the interests.

Table 1. Intra-day and inter-day accuracy and precision of the developed SEC-HPLC method. Data are mean ± SD, n = 3.

Concentration (µg/ml)	Calculated Concentration (µg/ml)	Accuracy (%)	RSD (%)*
Intra-Day
20	20.4 ± 0.9	102.2 ± 4.7	4.5 ± 2.7
50	50.0 ± 1.4	99.9 ± 2.9	2.0 ± 0.8
90	90.6 ± 1.3	100.6 ± 1.4	1.8 ± 0.5
Inter-day
20	19.8 ± 0.8	99.0 ± 4.1	4.8 ± 1.0
50	51.4 ± 2.5	102.8 ± 5.1	3.5 ± 1.5
90	89.7 ± 0.6	99.7 ± 0.7	1.2 ± 1.3

*RSD (%) = $\frac{\mathit{Population}\mathrm{ }\mathit{standard}\mathrm{ }\mathit{deviation}}{\mathit{Population}\mathrm{ }\mathit{mean}}\mathrm{ }x\mathrm{ }100$.

Table 2. Percentage of trastuzumab remaining after different stress conditions measured by SEC-HPLC.

	Trastuzumab remained (%)
Stress conditions	Solution at 0.21 mg/ml	Solution at 21 mg/ml
Mechanical stress
Sonication	97.3 ± 5.0	98.6 ± 6.3
Vortex	101.7 ± 1.0	100.1 ± 0.6
Freeze-and-thaw (7 cycles)	95.4 ± 3.3	102.9 ± 5.1
pH (4 °C, 16 h)
pH 2	68.6 ± 8.9****	63.4 ± 6.2****
pH 4	104.7 ± 2.9	118.3 ± 9.7*
pH 10	72.1 ± 5.4***	86.5 ± 6.5
pH 12	24.4 ± 2.8****	28.3 ± 6.2****
Temperature (time)
60 oC (24 h)	102.5 ± 8.4	136.7 ± 4.2****
60 oC (5 days)	90.4 ± 0.8	124.9 ± 8.3**
75 oC (24 h)	ND#	ND#

Two different trastuzumab concentrations (0.21 mg/ml and 21 mg/ml) were assessed as mean ± SD (n = 3).

^#ND: not detectable (LOD = 0.6 μg/ml); *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3. SDS-PAGE of trastuzumab solutions 0.21 mg/ml (L) and 21 mg/ml (H) after (A) mechanical stress. Trastuzumab control (C) of 21 mg/ml showed a single band at molecular weight of approximately 150 kDa. Sonication (S), vortexing (V) and repeated freeze-and-thaw (FT) did not show any aggregation or fragmentation; (B) solutions were adjusted to pH 2, 4, 10 and 12, and stored at 4 °C for 16 h or (C) incubated at 60 °C for 24 h or 5 days.

Figure 4. Stability of trastuzumab when stored at (A) -80 °C, (B) 4 °C, (C) 25 °C and (D) 37 °C for up to 12 months. Left: percentage of trastuzumab detected in 0.21 mg/ml (circle), 2.1 mg/ml (square) and 21 mg/ml (triangle) solutions by SEC-HPLC (n = 3, Mean ± SD); Right: SDS-PAGE at the end of 12 months storage.

Figure 5. Anti-proliferation activity of trastuzumab solutions of (A) 0.21 mg/ml and (B) 21 mg/ml stored at 4 °C after 6 and 12 months. Cell viability of HER2+ breast cancer cell BT-474 was measured by MTT assay after 5-day treatment (Mean ± SD, each concentration was measured in 4 wells, n = 3).