Investigating clearance mechanisms for recombinant activated factor VII in a perfused liver model

Abstract

Clearance mechanisms for recombinant activated human FVII (rFVIIa; NovoSeven), a heterogeneously glycosylated protein, have yet to be fully elucidated, but may involve the liver. The effects of the gamma-carboxy glutamic acid (Gla) domain and the sialic acid content of the protein on rFVIIa clearance were investigated following intravenous administration of rFVIIa lacking the Gla domain, des(1-44) rFVIIa and asialo-rFVIIa in pharmacokinetic (PK) studies and perfused rat livers. PK parameters for both rFVIIa and des(1-44) rFVIIa had similar biphasic clearance profiles, as well as half-lives ([t(1/2)]=80 and 88 minutes, respectively), while asialo-rFVIIa was cleared quickly (t(1/2)=21 minutes) with a linear clearance profile. Perfused liver studies with all proteins (10 nM) mirrored the trends in profiles observed in the PK study. rFVIIa and des(1-44) rFVIIa were cleared to a similar extent, 41% and 35%, respectively, after 1 h, whereas plasma-derived FVII from humans (which has a higher sialylation content than rFVIIa) was cleared to a lesser extent (21%). Asialo-rFVIIa, on the other hand, was almost totally cleared and when an excess of asialo-orosomucoid was added to the perfusate, its clearance was significantly reduced (by 34%) and also for rFVIIa, albeit to a lesser extent (by 14%). Together these data suggest that carbohydrate receptor(s) (e.g. the asialoglycoprotein receptor, ASGPR) play a role in asialo-rFVIIa and rFVIIa clearance. In vivo and liver clearance data correlated well showing similar trends and indicated that rFVIIa clearance is not affected by the Gla domain, but rather by a subpopulation of N-glycosylated structures on rFVIIa.

Recombinant Activated Factor VII Is Reabsorbed in Renal Proximal Tubules and Is a Ligand to Megalin and Cubilin

“…The inability of RAP to completely abolish endocytosis of 125 I-rFVIIa suggests that receptors other than megalin and cubilin may also be involved. ASGPR has been reported to be expressed in human proximal tubules [42] and is suggested to have a role in the clearance of rFVIIa in a perfused liver model [17]. However, it does not appear to play a role in endocytosis of rFVIIa in the OK cells as an excess of ASOR was unable to reduce endocytosis of 125 I-rFVIIa (fig.…”

“…We have reported that rFVIIa, following intravenous administration in rats, is cleared and distributed into endosomes and lysosomes within hepatocytes and sinusoidal Kupffer cells [16]. In an in situ perfused rat liver, at least one clearance mechanism has been suggested, involving clearance of a subpopulation of rFVIIa, based on the unsialylated N-terminal carbohydrate moieties [17]. To our knowledge, little data are available about the specific in vivo clearance mechanisms of injected rFVIIa in catabolic organs other than the liver.…”

Expression of recombinant human coagulation factors VII (rFVII) and IX (rFIX) in various cell types, glycosylation analysis, and pharmacokinetic comparison

“…Asialo rFVIIa clears quickest, whereas pdFVII, having a higher degree of sialylation, is cleared to a lesser extent [1]. In the case of FIX, the degrees of serine phosphorylation and tyrosine sulfation in the activation peptide have been postulated to influence pharmacokinetic behavior, especially in vivo recovery [2].…”

Glycoengineering of pertuzumab and its impact on the pharmacokinetic/pharmacodynamic properties

“…On the other hand, the impact of glycosylation on the PK behavior of mAbs was also investigated among these years, especially for terminal sialic acid. It is found that the sialylation degree of a glycoprotein can play a significant role in the serum half-life353637. Glycoproteins can be removed from the circulation via a sugar-based interaction with ASGPRs on hepatic cells373839.…”

“…Glycoproteins can be removed from the circulation via a sugar-based interaction with ASGPRs on hepatic cells373839. Several studies have reported that the ASGPRs induced endocytosis can lead to the clearance of glycoproteins in circulation364041. Sialic acid content varies depending on several factors including cell types and fermentation process, all these factors can result in different terminal sugar moieties of mAbs.…”