Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins

Abstract

Recombinant glycoprotein therapeutics produced in nonhuman mammalian cell lines and/or with animal serum are often modified with the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc; refs. 1,2). This documented contamination has generally been ignored in drug development because healthy individuals were not thought to react to Neu5Gc (ref. 2). However, recent findings indicate that all humans have Neu5Gc-specific antibodies, sometimes at high levels. Working with two monoclonal antibodies in clinical use, we demonstrate the presence of covalently bound Neu5Gc in cetuximab (Erbitux) but not panitumumab (Vectibix). Anti-Neu5Gc antibodies from healthy humans interact with cetuximab in a Neu5Gc-specific manner and generate immune complexes in vitro. Mice with a human-like defect in Neu5Gc synthesis generate antibodies to Neu5Gc after injection with cetuximab, and circulating anti-Neu5Gc antibodies can promote drug clearance. Finally, we show that the Neu5Gc content of cultured human and nonhuman cell lines and their secreted glycoproteins can be reduced by adding a human sialic acid to the culture medium. Our findings may be relevant to improving the half-life, efficacy and immunogenicity of glycoprotein therapeutics.

Figure 1. ELISA and Western-Blot Detection of Neu5Gc on Biotherapeutic Antibodies by Anti-Neu5Gc IgY Antibodies from Chickens or IgG Antibodies from Normal Human Serum

Cetuximab (Cet) and Panitumumab (Pan) were treated with active sialidase to eliminate Sia epitopes or with heat-inactivated sialidase as control. Samples were used for ELISA (A) or Western Blot (B), in which Neu5Gc was detected using an affinity-purified chicken anti-Neu5Gc IgY or control IgY. In an additional ELISA (C), Cet and Pan were used for coating, then blocked, and sialic acid epitopes eliminated chemically using sodium metaperiodate. The reaction was stopped with sodium borohydride. As a control, periodate and borohydride were pre-mixed and then added to the wells (the borohydride inactivates the periodate). ELISA samples were studied at least in triplicate and data shown are Mean +/- SD. ***p <0.001, Paired Two-tailed t-test. (D) Cet and Pan were treated with sialidase or heat-inactivated sialidase as in Figure 1A and used for coating ELISA wells, then blocked and incubated with human anti-Neu5Gc IgG that had been purified from the serum of healthy humans and biotinylated. Samples were studied in triplicate and data shown as Mean +/- SD. ***p <0.001 (E) Cet and Pan (1 μg each) were separated by SDS-PAGE and Coomassie stained or blotted (see Figure 1B). Neu5Gc content was detected by using biotinylated human anti-Neu5Gc IgG. (F) Immune complex formation with Cet or Pan in whole human serum was detected using the CIC (C1Q) ELISA Kit (Buehlmann) as described in the manufacturer's guidelines. The absorbance was measured at 405 nm. Samples were studied in triplicate and data are shown as Mean +/- SD. **p <0.01, Paired Two-tailed t-test. Gels in Panels B and E were cropped for clarity of presentation. Full-length blots/gels are presented in Supplementary Figures 1-4.

Figure 2. Effects of anti-Neu5Gc antibodies on the kinetics of therapeutic antibodies in mice with a human-like Neu5Gc-deficiency, levels of anti-Neu5Gc IgG in mice after injections of the therapeutic antibodies, and binding of IgG anti-Neu5Gc antibodies from whole human serum to Neu5Gc on the Fab fragment of Cetuximab

(A) Cmah null mice were first injected i.v. with the therapeutic antibodies (TAbs), namely Cetuximab (Cet) or Panitumumab (Pan), and mouse serum from Cmah null mice containing anti-Neu5Gc antibodies (or serum from naïve mice or control immunized mice) was then passively transferred by IP injection. Mice were bled periodically after the passive transfer of mouse serum. Concentration of Cet and Pan in the isolated sera was determined by Sandwich ELISA. Absorbance was measured at 495 nm. The Y axis starts at 60%, in order to better display the difference in kinetics. ***p <0.001, Unpaired Two-tailed t-test. (B) Cmah null mice were injected i.v. with Cet or Pan weekly and were bled initially, and after the 3rd i.v. injection. In order to detect Neu5Gc specific antibodies by ELISA, wells were coated with human (Neu5Gc-deficient) and chimpanzee (Neu5Gc-positive) serum glycoproteins (Upper Panel), or alternatively with human or bovine fibrinogen (Lower Panel). Data were obtained in triplicate. (C) Fab fragments of Cet and Pan were isolated using the Pierce® Fab Preparation Kit according to the manufacturer's manual. Fab fragments (1 μg/well) were used as target molecules in ELISA. Sialic acid specific binding was determined with sodium metaperiodate treatment. Wells were then blocked and incubated with human sera (S30 and S34 with low and high anti-Neu5Gc IgG titers, respectively, from Ref. 11). Binding of human IgG was detected by using anti-human IgG-Fc. The absorbance was measured at 490 nm and ELISA samples were studied in triplicate. *= p <0.05. Paired Two-tailed t-test.

Figure 3. An approach to reduce Neu5Gc Contamination in Biotherapeutic Products

(A-B) Human 293T cells were grown in the presence of 5 mM Neu5Gc for 3 days. The cells were then washed with PBS, split into two identical cultures, and 5 mM Neu5Ac was added to one of the cultures as shown on the graph. Cells were harvested as described in Methods, and the Neu5Gc and Neu5Ac content of both the ethanol soluble (A) and ethanol precipitable proteins (B) was analyzed by HPLC. The percent Neu5Gc shown is the amount of Neu5Gc relative to the total sialic acids. (C-E) Feeding of CHO cells with free Neu5Ac reduced Neu5Gc in the whole cell membranes and in secreted glycoproteins. Stably transfected CHO-KI cells expressing a recombinant soluble IgG-Fc fusion protein were grown in the absence or presence of 5 mM Neu5Ac. The individually collected media was centrifuged to remove cell debris and adjusted to 5 mM Tris-HCl pH 8. The fusion protein was purified using Protein-A Sepharose. (C) Sialic acid content was determined by DMB-HPLC analysis as described in Methods. The area under each peak was obtained and the percent of Neu5Gc in each sample was determined relative to Neu5Ac. (D) Total cell membranes from the same CHO cells were prepared and used for DMB-HPLC analysis. (E) CHO membrane proteins from the above experiments were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The expression of Neu5Gc was detected by incubating with polyclonal affinity purified chicken anti-Neu5Gc antibody, as described under Methods. This panel shows a blot of the full-length gel of the two relevant lanes.