Sugar Matters: Improving In Vivo Clearance Rate of Highly Glycosylated Recombinant Plasma Proteins for Therapeutic Use

Abstract

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. N-glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA₁₂₀) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.

Keywords: C1-inhibitor; glycosylation; recombinant protein.

Figure 1

Production levels of recombinant C1-inhibitor (rC1-INH) and half-life in vivo. (A) Antigen and activity levels of C1-INH produced by 57 different Chinese hamster ovary (CHO) cell clones. Antigen and activity levels correlate with a correlation efficiency of r = 0.962 p < 0.001 (Pearson two-tailed analysis). (B) Antigen and activity levels of rC1-INH in culture supernatants of the highest producing C1-INH clones after upscaling. (C) In vivo half-life of plasma-derived C1-INH (pdC1-INH) (Cetor) (n = 2), rC1-INH isolated from CHO clone 9 (n = 6) and rC1-INH isolated from CHO clone 17 (n = 2).

Figure 2

Terminal galactose screening for rC1-INH using RCA₁₂₀. (A) Inhibition of desialylated C1-INH to RCA₁₂₀ by pdC1-INH, rC1-INH and desialylated C1-INH as positive control. (B) Inhibition of desialylated C1-INH to RCA120 by rC1-INH, passed over an RCA120 column to remove terminal galactoses (rC1-INH RCA FT) and with rC1-INH eluted from the RCA120 column (rC1-INH RCA eluate). We can see that the capacity of inhibition by rC1-INH RCA FT is similar to pdC1-INH (dotted line), while the inhibitory capacity of rC1-INH RCA eluate is similar to desialylated C1-INH (solid line). Values are means ± SD of three independent experiments. * p < 0,05; **** p < 0,001 (according to two-tailed Students’ T-test).

Figure 3

N-glycan of rC1-INH, rC1-INH RCA FT and rC1-INH RCA eluate. N-glycans were analyzed by PGC nano-LC-ESI-MS/MS and visualized by extracted ion chromatograms (EIC). The N-glycosylation profile of rC1-INH RCA FT (B) shows a reduced relative abundance of terminal galactose and an increased relative abundance of terminal sialic acid compared to the N-glycosylation profile of rC1-INH (A). In contrast, rC1-INH eluate displays an increased abundance of terminal galactose and reduced abundance of terminal sialic acid (C). The top ten most abundant glycans are annotated per sample and assigned structural schemes. These spectra are representative of three different technical replicates. (D) Relative abundance of key structural motifs of rC1-INH and rC1-INH RCA₁₂₀ FT. Relative quantification and structural elucidation of the 40 most abundant glycans was performed per sample (see Supplemental Figure S2). For the relative abundances of the glycan motifs, terminal galactose, terminal GlcNAc and terminal sialic acid, the relative abundance of glycans carrying these motifs were multiplied by the number of these motifs per glycan and summed. Analyses were performed in triplicate and the standard deviation (SD) is given.

Figure 4

Function and half-life of rC1-INH without terminal galactoses. The association rate constants k_on (M⁻¹s⁻¹) for the inhibition of C1s (A), Kallikrein (B), Plasmin (C) and (D) FXIa by pdC1-INH, rC1-INH and rC1-INH RCA FT were determined. Values are means ± SD of three independent experiments, each performed in duplicate. No significant differences were observed. (E) The half-lives of pdC1-INH (n = 2), rC1-INH (n = 3) and rC1-INH RCA FT (n = 2) show that the half-lives of pdC1-INH and rC1-INH RCA FT are similar, while the half-life of rC1-INH is clearly reduced.