Characterization of differences in isoelectric focusing behavior of α1‐proteinase inhibitor products

In Fall 2004, the FDA was notified that a plasma-derived α1-proteinase inhibitor (α1-PI) product, Aralast manufactured by Baxter and used in augmentation therapy of congenital α1-PI (also known as α1-antitrypsin) deficiency, shows an isoelectric focusing (IEF) pattern different from that of α1-PI in normal plasma. An α1-PI band shift suggested that most of the α1-PI in Aralast is modified and carries an additional negative charge. In January 2005, a patient advocacy group, the Alpha-1 Foundation, posted a letter on their Web site informing patients of this observation and potential consequences that may impact product safety, e.g., altered terminal half-life, altered biodistribution, increased antigenicity, and organ toxicity. The Alpha-1 Foundation advised patients to discuss these observations concerning Aralast with their physicians. In response to these reports of an aberrant IEF pattern of Aralast, we investigated the IEF behavior of all licensed α1-PI products: Prolastin (Talecris Biotherapeutics, Research Triangle Park, NC), Aralast (Baxter Healthcare Corporation, Deerfield, IL), and Zemaira (ZLB Behring, King of Prussia, PA). These products were licensed in 1987, 2002, and 2003, respectively, and approval was related to the ability of a weekly intravenous dose (60 mg of functional α1-PI per kg of body mass) to maintain circulating α1-PI levels of at least 11 μmol per L (measured antigenically) and to acceptable safety profiles. The FDA continues to review reported adverse events for all three products. α1-PI deficiency is treated by lifelong augmentation therapy; therefore, understanding the nature of any biochemical differences between α1-PI in products and α1-PI in plasma and monitoring the safety of long-term product administration are important. IEF is an electrophoretic method that separates protein species with different isoelectric points (pI’s). Although used to phenotype patients, IEF has not been required for characterization of plasma-derived products. On IEF gels, α1-PI separates into a number of bands due to sequence and oligosaccharide heterogeneity. In plasma, two major α1-PI glycoforms with three biantennary N-glycans (M6) and with two biantennary and one triantennary N-glycans (M4) give rise to bands M6 and M4, respectively (Fig. 1, Lane 1). Each N-glycan branch is capped with sialic acid; thus, glycoform M4 is more negative than M6 by one charge. The IEF pattern of each product also shows two major bands (Fig. 1, Lanes 2-4). These bands in Prolastin (Fig. 1, Lane 4) migrate like bands M6 and M4 of plasma α1-PI (Fig. 1, Lane 1). In Zemaira (Fig. 1, Lane 3), major bands are slightly shifted from positions M6 and M4 toward the anode. In Aralast (Fig. 1, Lane 2), approximately twothirds of forms M6 and M4 appear to carry an additional negative charge and focus at positions M4 and M2, respectively. Each product appears to consist of two populations of α1-PI: one population giving rise to bands similar to M6 and M4 from plasma and constituting the major population in Prolastin and Zemaira, but a minor population in Aralast, and another population carrying an additional negative charge, having forms M6 and M4 shifted to positions M4 and M2, respectively, and comprising the major component of Aralast, but a minor component of both Prolastin and Zemaira. To investigate sequence heterogeneity, N-glycan heterogeneity was eliminated by removal of sialic acid with neuraminidase (EC 3.2.1.18) from Clostridium perfringens. IEF analysis of desialylated samples confirmed that each product contains two major populations of α1-PI corresponding to two major bands (Fig. 1, Lanes 5-7); increased pI values result from removal of sialic acid. The most intense band of each product after desialylation corresponds to the two major bands observed for each prodMay 2006465872Letters to the EditorLETTERS TO THE EDITORLETTERS TO THE EDITOR