Charge variants

The EP limits charged variants of somatropin, e.g., deamidated forms, hy a CZE method that is characterized hy an ammonium phosphate buffer at pH 6.0 as an electrolyte, an uncoated fused silica capillary, and a UV detection at 200 nm (see Eigure 4 for robustness see reference 43). 

Isoelectric focusing (lEF) is another routine slab gel technique for the identification of biotechnology products [37]. The isoelectric points (pi) of the produced protein and its variants can be monitored. The presence of several charge variants is a common feature of recombinant antibodies and can be the result of, for example, deamidation or differences in processing of G-terminal lysine residues [38, 39]. The consistency of the charge heterogeneity can be monitored by using lEF. A typical lEF analysis is shown in Fig. 3.21 B. The pi values of the protein isoforms can be identified for aU of the samples. The lEF patterns for antibodies produced in batch, fed-batch and perfusion modes with PER.G6 cells are very similar. 

We know from the standard model of elementary particle physics [116] that there is a tiny weak interaction contribution to every Coulomb interaction. For ordinary matter, where particle interconversion can be ignored, weak interactions due to exchange of neutral Z vector bosons are involved. Unlike the Coulomb interaction, the (neutral and charged variants of) weak interactions do not conserve parity. This leads, in consequence, to a very small energy difference between mirror-image molecules (enantiomers), which in turn might prove to be of importance for the development of a homochiral biochemistry on our planet [117]. 

CIEF is an important technique for analyzing the charged variants of EPO glycoforms on the basis of sialic acid content differences. Cifuentes et al. showed that EPO glycoforms could be resolved by using a mixture of broad and narrow... 

Lutter, P., Meyer, H.E., Langer, M., Witthohn, K., Dormeyer, W., Sickmarm, A., and Bluggel, M., 2001, Investigation of charge variants of rViscumin by two-dimensional gel electrophoresis and mass spectrometry. Electrophoresis 22 2888-2897. 

M.F. Ebersold and A.L. Zydney, Separation of protein charge variants by ultrafiltration. Biotech. Prog. 20 (2004) 543-549. 

Khawli, L.A., Goswami, S., Hutchinson, R., et al. (2010) Charge variants in IgGl Isolation, characterization, in vitro binding properties and pharmacokinetics in rats. mAbs, 2 (6), 613-624. 

Japanese Pharmacopoeia (JP), and the EP. In addition, a CZE method is currently used as an identification test for EPO concentrated solution in the EP. This represents the first example of the use of CE for the monitoring of a biopharmaceutical by an official method. A second example is in the final stage of implementation and involves the determination by CZE of charge variants in somatropin preparations. This test, which was found to provide more precise quantitative data, will replace the current isoelectric focusing method. 

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