Table 3.
CIEF and microchip IEF (mIEF)]
| Analyte | Mode | Capillary coating | Capillary | Detector | Ampholyte | Catholyte | Anolyte | Notes | Ref |
|---|---|---|---|---|---|---|---|---|---|
| In-house EPO, Fc-fusion protein, and IgG |
iCIEF | Fluorocarbon (Protein Simple) |
iCE280 Analyzer; 50 mm, 100 µm id |
UV 280 nm | Pharmalyte pH 3–10, 4–6.5, 5–8, and 8–10.5 |
0.1 M NaOH in 0.1% MC |
0.08 M phosphoric acid in 0.1% MC |
Wide range of Therapeutic protein applications |
[51] |
| In-house noninfectious virus-like particles |
iCIEF | Fluorocarbon (Protein Simple) |
iCE280 Analyzer; 50 mm, 100 µm id |
UV 280 nm | Pharmalyte pH 2.5–5 and 3–10 |
0.1 M NaOH in 0.1% MC |
0.08 M phosphoric acid in 0.1% MC |
Charge characterization of virus-like particles |
[52] |
| In-house vaccine carrier protein |
iCIEF | Fluorocarbon (Protein Simple) |
iCE280 Analyzer; 50 mm, 100 µm id |
UV 280 nm | Pharmalyte pH 3–10 and 4–6.5 |
0.1 M NaOH in 0.1% MC |
0.08 M phosphoric acid in 0.1% MC |
Characterization of polysaccharide vaccine carrier protein |
[53] |
| In-house IgG2κ mAb |
CIEF | PDMA | 180 mm, 50 µm id |
LIF ex. 543.5 nm / em. 590 nm |
Pharmalyte pH 3–10, 0.001% BSA |
20 mM NaOH | 20 mM phosphoric acid |
Determination of deamidation rates in mAb |
[54] |
| Trypsinogen, β-lactoglobulin, BSA, and ovalbumin |
CIEF | 4% acrylamide 0.6% cross- linker |
Various capillary lengths and id |
LIF ex. 488 nm | BioRad pH 3–10 | 20 mM NaOH | 10 mM phosphoric acid |
Microchip interface for 2D CIEF and CGE separations |
[55] |
| β-lactoglobulin A, hemoglobin A, myoglobin, α-chymotrypsinogen A, ribonulcease A, cytochrome c, lysozyme |
CIEF | Seven PEEK tubing segments, 1.55 cm, 395 µm id connected by Nafion membrane 0.2 cm, 330 µm id |
TOF and Orbitrap | Various MS-compatable carrier ampholytes |
Various electrolytes at each Nafion junction modified the local pH of the carrier ampholyte |
Segmented capillary for selective mobilization |
[60] | ||
| Insulin receptor and protein tryptic digests |
CIEF | LPA | 50 cm, 50 µm id | Sheath flow ESI- Orbitrap-MS |
Glutamate, asparagine, glycine, proline, histidine, and lysine |
0.1% Formic acid, pH 2.5 |
0.3% ammonium hydroxide, pH 11 |
Amino acids used as low MW ampholyte |
[64] |
| In-house IgG mAb |
iCIEF | Proprietary photoreactive layer |
12-channel cartidge; 50 mm, 100 µm id |
Chemiluminescence from secondary antibody |
Pharmalyte pH 5–8 (30%) and pH 8–10.5 (70%) |
0.1 M NaOH in 0.1% MC |
0.08 M phosphoric acid in 0.1% MC |
CIEF with immunoassay detection |
[65] |
| Donated mAb products | imIEF | Uncoated quartz | MCE-2010 system, 2.7 cm |
UV 280 nm | Protein Simple pH 3–10, 5–8, and 8–10.5 |
300 mM NaOH, 0.4% HPMC |
200 mM phosphoric acid, 0.4 % HPMC |
mIEF of mAb charge variants |
[141] |
| Model Proteins | iMIF | 100 µm id | Immunoblot | Polyprotic carboxylic amino acids |
20 mM lysine | 20 mM arginine | Integrated microchip for separation and immunoblot |
[142] | |
Capillary: actual length, inner diameter. Bovine serum albumin (BSA), Linear polyacrylamide (LPA), Methylcellulose (MC), Polydimethylacrylamide (PDMA), Polyvinyl alcohol (PVA),