ABSTRACrPolygacturonse (PG) The ripening of tomato fruit is characterized by a series of coordinated biochemical and physiological changes which collectively contribute to the final texture, color, and flavor ofthe ripe fruit. The softening of tomato fruit during ripening has been studied extensively during the past 20 years and a large body of evidence has accumulated which suggests the cell wall enzyme PG2 plays a major role in this process. Several lines of evidence which support this include: (a) a rough correlation between levels of extractable PG activity and the rate of softening for a number of tomato cultivars (6, 13); (b) a lack of correlation between the activity of other cell wall enzymes (e.g. cellulase and A3-1,3-glucanase) and the rate of fruit softening (14, 29); (c) similarities between in vivo cell wall degradation products during ripening and in vitro cell wall products released by the action of purified PG on isolated fruit cell walls (15,30); and (d) the demonstration that slow ripening cultivars and nonripening tomato mutants are deficient in PG activity (6,22,28). Although these lines of evidence suggest a major role for PG in softening, other factors such as the levels of free and bound Ca2" in the cell wall and substrate accessibility may also be important (7).Recently, several groups have studied changes in gene expres- ' Supported by gifts from Chesebrough-

BackgroundBAY 81–8973 (Kovaltry) is an unmodified full-length recombinant factor VIII (rFVIII) for treatment of hemophilia A. The BAY 81–8973 manufacturing process results in a product of enhanced purity with a consistently high degree of branching and sialylation of N-linked glycans. This study evaluated whether a relationship exists between N-linked glycosylation patterns of BAY 81–8973 and two other rFVIII (sucrose-formulated rFVIII [rFVIII-FS; Kogenate FS]) and antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM; Advate) and their pharmacokinetic (PK) characteristics.Materials and methodsN-linked glycans or terminal carbohydrates were enzymatically removed from immobilized BAY 81–8973, rFVIII-FS, and rAHF-PFM proteins and analyzed using high-performance liquid chromatography to determine the percentage of individual N-linked glycan structures and degree of sialylation of each structure. PK data were available from two separate phase 1 crossover studies in which the PK profile of BAY 81–8973 was compared with that of rFVIII-FS (n=26) and rAHF-PFM (n=18) in patients with severe hemophilia A who received a single 50 IU/kg dose of each product.ResultsBAY 81–8973 and rFVIII-FS had increased N-linked glycan branching with higher levels of sialylation compared with rAHF-PFM. Levels of trisialylated glycans were 29.0% for BAY 81–8973 vs 11.5% for rFVIII-FS and 4.8%–5.5% for rAHF-PFM; tetrasialylated glycans were 12.0% vs 2.8% and 0.6%, respectively. Degree of sialylation was 96% for BAY 81–8973, 94% for rFVIII-FS, and 78%–81% for rAHF-PFM. Based on chromogenic assay results from the single-dose phase 1 PK studies, BAY 81–8973 half-life was 15% longer than that for rFVIII-FS and 16% longer than rAHF-PFM.ConclusionIncreased N-glycan branching and sialylation were seen for BAY 81–8973 vs rFVIII-FS and rAHF-PFM. Improved PK for BAY 81–8973 relative to rFVIII-FS and rAHF-PFM as seen in single-dose crossover PK studies might be related to this greater level of branching and sialylation, which can prolong the time BAY 81–8973 remains in the circulation.

The circulatory half-life of recombinant factor VIII (rFVIII) products is affected by glycosylation of the FVIII protein, including N-linked glycan branching and terminal sialic acid occupancy, primarily through receptor-mediated hepatic clearance (eg, asialoglycoprotein receptor [ASGPR] and lipoprotein receptor-related protein [LRP]). BAY 81-8973 (Kovaltry®, Bayer, Berkeley, CA) is an unmodified full-length rFVIII for treatment of hemophilia A. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of branching and sialylation of N-linked glycans. This study evaluated whether a relationship exists between N-linked glycosylation patterns and pharmacokinetic (PK) characteristics of BAY 81-8973 and 2 other rFVIII products (sucrose-formulated rFVIII [rFVIII-FS; Kogenate® FS, Bayer] and antihemophilic factor (recombinant) plasma/albumin-free method [rAHF-PFM; Advate®, Shire, Westlake Village, CA]). N-linked glycans or terminal carbohydrates were enzymatically removed from immobilized BAY 81-8973, rFVIII-FS, and rAHF-PFM proteins and analyzed using high-performance liquid chromatography to determine the percentage of individual N-linked glycan structures and degree of sialylation of each structure. PK data were available from 2 separate phase 1 crossover studies in which the PK profile of BAY 81-8973 was compared with that of rFVIII-FS (n=26) and rAHF-PFM (n=18) in patients with severe hemophilia A who received a single 50-IU/kg dose of each product. BAY 81-8973 and rFVIII-FS had increased N-linked glycan branching with higher levels of sialylation compared with rAHF-PFM. Levels of trisialylated glycans were 29.0% for BAY 81-8973 versus 11.5% for rFVIII-FS and 4.8% to 5.5% for rAHF-PFM; tetrasialylated glycans were 12.0% versus 2.8% and 0.6%, respectively. Degree of sialylation was 96% for BAY 81-8973, 94% for rFVIII-FS, and 78% to 81% for rAHF-PFM. Based on chromogenic assay results from the single-dose phase 1 PK studies, BAY 81-8973 half-life was 15% longer than that for rFVIII-FS and 16% longer than rAHF-PFM. Increases in the percentage of sialylated tri-antennary and tetra-antennary N-glycans correlated well with longer half-life of rFVIII in humans (adjusted R2=0.978 and 0.892 for tri-antennary and tetra-antennary N-glycans, respectively). Higher percentages of sialylation (ie, sialic acid capping) correlated with a longer half-life (adjusted R2=0.697), but the relationship was not as strong as that between glycan branching and half-life. Improved PK for BAY 81-8973 relative to rFVIII-FS and rAHF-PFM as seen in single-dose crossover PK studies might be related to this greater level of branching and sialylation, which may prolong the time BAY 81-8973 remains in the circulation. Disclosures Teare: Bayer: Employment. Kates:Bayer: Employment. Shah:Bayer: Employment. Garger:Bayer: Employment.

As an increasing number of recombinant therapeutic glycoproteins are manufactured and investigated, the importance of their attached glycans is becoming more widely reported and understood. Regulatory agencies expect detailed "extended characterization" of the glycoprotein as well as routine, well-controlled "release assays" with specifications to be employed for quality control of each manufactured lot. In this chapter we will briefly discuss relevant glycan issues in the area of therapeutic recombinant glycoprotein manufacture and describe in detail two assays that are employed in the development of, for example, recombinant Factor VIII for the treatment of hemophilia.