Pompe's disease is caused by a deficiency of the lysosomal enzyme acid ␣-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and Nglycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78 -113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204 -782) contains the 3.9-and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.Lysosomal acid ␣-glucosidase (GAA 1 ; EC 3.2.1.3) is an exo-1,4-and -1,6-␣-glucosidase that hydrolyzes glycogen to glucose. The cDNA for GAA encodes a protein of 952 amino acids with a predicted molecular mass of 105 kDa (1). The newly synthesized precursor has an amino-terminal signal peptide for cotranslational transport into the lumen of the endoplasmic reticulum, where it is N-glycosylated at seven glycosylation sites, resulting in a glycosylated precursor with an apparent molecular mass of 110 kDa.The intracellular processing of GAA has been investigated previously (2, 3). It was proposed that, after transport through the Golgi complex and targeting to the endosome/lysosome, the 110-kDa precursor is proteolytically processed at the amino terminus, resulting in a 95-kDa intermediate with a sequence beginning at amino acid 122. Prior to this study, the 95-kDa intermediate was proposed to be proteolytically processed to a 76-kDa form, which was believed to occur between amino acids 816 and 881 (3). The 76-kDa form is then proteolytically processed at the amino terminus at amino acid 204 to give the 70-kDa mature form (3). The nomenclature used for the processed forms of GAA is based on apparent molecular mass as determined by SDS-PAGE.The identities of the proteases involved in the maturation of GAA have never been established. GAA has been purified from many different tissues such as bovine testis (4), rat liver (5), pig liver (6), human liver (7), rabbit mus...

The amino acid sequences of ananain (EC3.4.22.31) and stem bromelain (3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. We present here the complete amino acid sequence of ananain and compare it with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya and actinidin from kiwifruit. Ananain is comprised of 216 residues with a theoretical mass of 23464 Da. This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain.

Figure 1. Illustration of subunit mispairing in trispecific antibodies with CODV (Crossover Dual Variable) IgG-like design. Article pubs.acs.org/ac

The manufacturing of virus occurs at a modest scale in comparison to many therapeutic proteins mainly because a gene therapy dose is typically only µg of vector. Although modest in scale the generation of high purity virus is challenging due to low viral expression levels and the difficulties in adequately characterizing such a large and complex molecule. A 100 L bioreactor might produce only 100 mg of virus that must be separated from host and process impurities that are typically greater by several orders of magnitude. Furthermore, in the later purification stages the main milieu component is often virus at low concentration (µg/mL) which may non-specifically adsorb to purification surfaces resulting in a lowered virus recovery. This study describes our approach to develop a scalable, manufacturable robust process for an Adenovirus (Ad) gene therapy vector. A number of analytical tools were developed to guide the purification design. During process development, two human proteins, SET and nucleolin, were identified in viral preparations. To our knowledge, this is the first time that SET and nucleolin have been described in Ad. In this report we detail a process for their removal and the robust removal of all process, product and host cell impurities.