Analysis of Infliximab by Maurice
Introduction
Infliximab targets tumor necrosis factor alpha (TNFα) and has been shown to be effective in the treatment of autoimmune diseases like Crohn's disease1. Several biosimilars have already been approved2.
Maurice icIEF Method
Carboxypeptidase B (CpB) treatment: Infliximab was diluted to 1.0 mg/mL in water prior to CpB digestion. CpB (1 mg/mL stock solution) was added at a ratio of 1:100 (CpB to sample) and incubated at 37 °C for 20 minutes and then placed on ice. CpB was obtained from Sigma-Aldrich (PN C9584).
Sample preparation: Infliximab was diluted to 0.2 mg/mL in the ampholyte solution.
Ampholyte solution: Pharmalytes 8–10.5 (3%) and 5–8 (1%) containing 3.2 M urea, 5 mM IDA and10 mM arginine.
pI markers: 5.85 and 10.1.
Running conditions: 1 minute at 1500 V, then 8 minutes at 3000 V.
Imaging: Absorbance and fluorescence.
Results
icIEF analysis of infliximab is shown in Figure 1 with absorbance detection and in Figure 2 with fluorescence detection. Treatment with CpB the revealed presence of terminal lysine variants.
Figure 1. icIEF absorbance (top) and peak area percentages (bottom) of iInfliximab.
| Sample | Acidic Region | Main Peak | Basic Region | Δ Basic Region | |
|---|---|---|---|---|---|
|
Infliximab |
No treatment | 19.0 | 35.4 | 45.6 | N/A |
| CpB-treated | 39.9 | 57.5 | 2.6 | -4.3 | |
Figure 2. icIEF fluorescence (top) and peak area percentages (bottom) of infliximab.
| Sample | Acidic Region | Main Peak | Basic Region | Δ Basic Region | |
|---|---|---|---|---|---|
|
Infliximab |
No treatment | 20.8 | 33.1 | 46.2 | N/A |
| CpB-treated | 45.8 | 50.9 | 3.3 | -42.9 | |
Maurice CE-SDS Method
Sample preparation: Infliximab was diluted to 1 mg/mL with 1X Sample Buffer prior to treatment for 10 minutes at 70 °C in the presence of either 11.5 mM IAM (nonreducing) or 650 nM β-ME (reducing).
Running conditions: Samples were injected for 20 seconds at 4600 V, followed by a 25-minute separation (reducing) or a 35-minute separation (non-reducing) at 5750 V.
Results
Infliximab was analyzed on the CE-SDS platform method described above under reducing (Figure 3) and nonreducing (Figure 4) conditions, revealing the purity of the sample.
Figure 3. CE-SDS reduced (top) and peak area percentages (bottom) of infliximab. (IS) Internal standard. (LC) Light chain. (NGHC) Nonglycosylated heavy chain. (HC) Heavy chain.
| Sample | Other | NG | Intact |
|---|---|---|---|
| Infliximab | 3.7 | 0.0 | 96.3 |
Figure 4. CE-SDS non-reduced (top) and peak area percentages (bottom) of infliximab. (IS) Internal standard. (NG) Non-glycosylated.
| Sample | LC | NGHC | HC | Other |
|---|---|---|---|---|
| Infliximab | 30.9 | 0.6 | 68.2 | 0.4 |
References
- Remicade® (infliximab): 20 years of contributions to science and medicine, R Melsheimer, A Geldhof, I Apaolaza and T Schaible, Biologics: Targets and Therapy, 2019; 13:139–178.
- Biosimilars already approved and in development, T Dörner, J Isaacs, J Gonçalves, V Azevedo, G CastañedaHernández, R Strohal and I McInnes, Considerations in Medicine, 2017; 1:7–12.