Immune globulin subcutaneous, human 20% solution (Xembify®), a new high concentration immunoglobulin product for subcutaneous administration.

Immune globulin subcutaneous, human 20% solution (IGSC-C 20%, Xembify®)-a new 20% immunoglobulin (IgG) liquid product for subcutaneous (SC) administration-has been developed by Grifols. The IGSC-C 20% formulation is based on knowledge acquired from the formulation of Immune Globulin Injection (Human),10% Caprylate/Chromatography Purified (IGIV-C 10%, Gamunex®-C). The protein concentration was increased from 10% to 20% to provide a smaller volume for SC administration. The IGSC-C 20% manufacturing process employs the same caprylate/chromatography purification steps as IGIV-C 10%, with the addition of an ultrafiltration step so that the product can be formulated at a higher protein concentration. IGSC-C 20% has been produced at full industrial scale to support clinical studies and licensure. These batches were characterized using a comprehensive panel of analytical testing. The new IGSC-C 20% product maintains the same composition, neutralizing activity, purity, and quality characteristics found in IGIV-C 10%.

Production and purification of TAL6003: a novel version of the protease plasmin.

TAL6003 is an engineered form of human plasmin under development for thrombolytic therapy. TAL6003 (38.2kDa) contains nine disulfide bonds distributed within and between its two functional domains, a 25.2 kDa serine protease domain linked to a 13.0 kDa kringle I domain; kringles 2-5 present in native human plasmin have been deleted from this plasmin molecule. TAL6003 is expressed as its zymogen in Escherichia coli and is harvested in inclusion bodies. Following solubilization of inclusion bodies with urea as the chaotrope and glutathione as the reducing agent, this zymogen is refolded by dilution to a final concentration of 0.5mg/ml, with a yield of 48% (relative to total zymogen). Refolded TAL6003 zymogen is filtered, diafiltered, and filtered again prior to capture and purification on SP Sepharose, which is highly effective in removing host-cell protein. Subsequent affinity purification on ECH-Lysine Sepharose serves to capture polypeptide chains containing correctly refolded kringle 1 domain, which is the locus of the molecule's lysine-binding site, and to further eliminate host-cell protein. TAL6003 zymogen eluted from the ECH-Lysine Sepharose column is activated to TAL6003 with streptokinase, with an activity yield of approximately 80%. Proteolytically active TAL6003 is stripped of streptokinase by passage through an anion-exchange (Q) membrane and is then affinity purified on Benzamidine Sepharose, which serves to remove unreacted TAL6003 zymogen and proteolytically degraded TAL6003. An ultrafiltration/diafiltration step, to concentrate and to formulate TAL6003, completes the purification process. The final product exhibited a specific activity of close to unity and high purity by several relevant criteria.

Estrogen response element and the promoter context of the human and mouse lactoferrin genes influence estrogen receptor alpha-mediated transactivation activity in mammary gland cells.

A critical step in estrogen action is the recognition of estrogen responsive elements (EREs) by liganded estrogen receptor. Our current studies were designed to determine whether an extended estrogen response element half-site (ERRE) contributes to the differential estrogen responses of the human and mouse lactoferrin overlapping chicken ovalbumin upstream promoter/ERE sequences (estrogen response modules, ERMs) in the context of their natural promoters. Transient transfections of MCF-7 cells show that liganded estrogen receptor alpha (ERalpha) activates transcription of the human lactoferrin ERM fourfold higher than the mouse lactoferrin ERM in the context of their natural promoters. Since the ERRE of the human lactoferrin gene naturally occurs 18 bp upstream from the ERM and is absent in the mouse lactoferrin gene promoter, we created a chimeric mouse lactoferrin CAT reporter, which now encodes the ERRE in the identical location as in the human lactoferrin gene. The addition of the ERRE in the mouse lactoferrin gene rendered this reporter extremely responsive to estrogen stimulation. Using limited protease digestions and electrophoretic mobility shift assays, we showed that the binding and protease sensitivity of ERalpha bound to the mouse ERM with or without the ERRE, differed. Importantly, occupancy of additional nuclear receptors at the ERRE may contribute to ERalpha binding and activation. Furthermore, the presence of ERRE influences the selectivity of coactivators in liganded ERalpha-mediated transcriptional activity. When the receptor is bound to human and mouse plus genes, which contain the ERRE, steroid receptor coactivator (SRC)-2 was preferred, while SRC-1 and SRC-3 coactivators selectively enhanced the mouse lactoferrin gene activity. Moreover, peroxisome proliferator activated receptor-gamma coactivator-1 (PGC-1alpha) and PGC-1-related estrogen receptor coactivator (PERC) robustly increase the transcriptional function of ERalpha in the presence of the ERRE. In conclusion, these data show that the context of the lactoferrin gene influences the ERalpha-mediated transcriptional activity.