Performance comparison of protein A affinity resins for the purification of monoclonal antibodies.

During the selection of protein A affinity resin for the purification of monoclonal antibodies, dynamic binding capacity (Q(dyn10%)), volumetric production rate (Pr(vol)) and 'process robustness' are essential parameters to be evaluated. In this article, empirical mathematical models describe these parameters as a function of antibody concentration in load (C0), load flow rate (u(load)) and bed height (L). These models allow us to select optimal process conditions for each of the evaluated protein A affinity resins. C0, u(load) and L largely affect dynamic binding capacity (Q(dyn10%)) and volumetric production rate (Pr(vol)). Maximum Q(dyn10%) is generally obtained at high C0 and at low u(load). Maximum Pr(vol) is obtained at high C0 and at lowest L, run at high u(load). All evaluated resins have a relatively high robustness against variations in C0. |DeltaQ(dyn10%)/deltaC0| ranges from 0.0 to 7.8. It is clear that Q(dyn10%), Pr(vol) and 'process robustness' cannot be maximized all at the same time. Furthermore, some other aspects like IgG recovery, protein A leaching, easiness to pack, easiness to clean, number of re-uses and cost of production might be important to be taken into the equation. Certain evaluation parameters may be more important than others, depending on the specific situation. Therefore, a case-by-case evaluation is recommended.

A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest.

The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at 99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was

Monoclonal antibodies against metallothioneins from the human liver.

Six hybridomas secreting monoclonal antibodies directed against human fetal liver metallothioneins (MTs) have been generated and characterized. One antibody, K5A6, was specific for MT-1, the others recognized all isoMTs present in the human fetal liver. Each of these antibodies showed a unique cross-reactivity pattern when tested with MTs from the livers of different mammals. A double-antibody sandwich enzyme-linked-immunosorbent-assay (ELISA) was developed with the antibodies L2E3 and L5G2. This assay allows the detection of 60 pg human fetal liver MT and exhibits a metal dependent response for Zn-, Cd- and Hg-MT.

Identification of Metallothionein- and parathyroid hormone-related peptide (PTHrP)-positive cells in salivary gland tumours.

Ductal basal cells and myoepithelial cells (MEC) of normal salivary gland share metallothionein (MT)-positivity, while PTHrP positivity is restricted to ductal basal cells. We studied 21 benign and 4 malignant tumours in which MEC are thought to play a role using immuno-histochemical methods for detecting the presence of MT and PTHrP positive cells. In benign tumours, a shared positivity for MT and PTHrP is found in the inner layer of tubulo-ductal and trabecular structures, in part of the cells in the myxoid and chondroid matrices of pleomorphic adenoma, and in the basal epithelial lining of Warthin's tumours. In myoepithelioma almost all tumour cells demonstrate MT reactivity and a restricted positivity for PTHrP. MT-positive cells in oncocytoma were demonstrated in the periphery of some oncocytic islets, while PTHrP positivity was restricted to a few oncocytic cells. In malignant tumours, positivity for MT is found in the periphery of epithelial clusters of mucoepidermoid carcinomas, while PTHrP-positive cells are seen in cyst-like structures and scattered cells in solid arrangements of squamous cells. Although the biologic significance of the presence of MT in neoplastic cells is not yet clearly understood, MT may be necessary for the growth and differentiation in actively growing cells. The variability of MT expression in salivary gland tumours could be a reflection of the morphological heterogeneity and correlate with the degree of differentiation and maturation of the tumour cells. The observations suggest that MT may be considered an oncodevelopmental product.

Agenesis of corpus callosum and anophthalmia in the asplenia syndrome. A recognisable association?

We present a newborn infant with the asplenia syndrome and unique associated features of corpus callosum agenesis, anophthalmia and coloboma. Previous reports of eye abnormalities or corpus callosum agenesis in patients with asplenia suggest that this may represent a distinct clinically recognisable entity of abnormal lateralisation.