Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins

Chinese hamster ovary (CHO) cells are widely used for the manufacture of biotherapeutics, in part because of their ability to produce proteins with desirable properties, including 'human-like' glycosylation profiles. For biotherapeutics production, control of glycosylation is critical because it has a profound effect on protein function, including half-life and efficacy. Additionally, specific glycan structures may adversely affect their safety profile. For example, the terminal galactose-α-1,3-galactose (α-Gal) antigen can react with circulating anti α-Gal antibodies present in most individuals. It is now understood that murine cell lines, such as SP2 or NSO, typical manufacturing cell lines for biotherapeutics, contain the necessary biosynthetic machinery to produce proteins containing α-Gal epitopes. Furthermore, the majority of adverse clinical events associated with an induced IgE-mediated anaphylaxis response in patients treated with the commercial antibody Erbitux (cetuximab) manufactured in a murine myeloma cell line have been attributed to the presence of the α-Gal moiety. Even so, it is generally accepted that CHO cells lack the biosynthetic machinery to synthesize glycoproteins with α-Gal antigens. Contrary to this assumption, we report here the identification of the CHO ortholog of N-acetyllactosaminide 3-α-galactosyltransferase-1, which is responsible for the synthesis of the α-Gal epitope. We find that the enzyme product of this CHO gene is active and that glycosylated protein products produced in CHO contain the signature α-Gal antigen because of the action of this enzyme. Furthermore, characterizing the commercial therapeutic protein abatacept (Orencia) manufactured in CHO cell lines, we also identified the presence of α-Gal. Finally, we find that the presence of the α-Gal epitope likely arises during clonal selection because different subclonal populations from the same parental cell line differ in their expression of this gene. Although the specific levels of α-Gal required to trigger anaphylaxis reactions are not known and are likely product specific, the fact that humans contain high levels of circulating anti-α-Gal antibodies suggests that minimizing (or at least controlling) the levels of these epitopes during biotherapeutics development may be beneficial to patients. Furthermore, the approaches described here to monitor α-Gal levels may prove useful in industry for the surveillance and control of α-Gal levels during protein manufacture.National Center for Research Resources (U.S.) (Grant P41 RR018501-01

A boost of positive affect: the perks of sharing positive experiences

In a series of five studies we examined the relationship between sharing positive experiences and positive affect using a diary method (Study 1) and laboratory manipulations (Studies 2 and 3). All of these studies demonstrated that sharing the positive experience heightened its impact on positive affect. In Study 4, we conducted a four-week journal study in which the experimental participants kept a journal of grateful experiences and shared them with a partner twice a week. Control participants either kept a journal of grateful experience (without sharing), or kept a journal of class learnings and shared it with a partner. Those who shared their positive experiences increased in positive affect, happiness, and life satisfaction over the course of four weeks. Study 5 showed that those who received an “active-constructive” response to good news (enthusiastic support) expressed more positive affect than participants in all other conditions, indicating that the response of the listener is important. In sum, our findings suggest that positive affect, happiness, and life satisfaction reach a peak only when participants share their positive experiences and when the relationship partner provides an active-constructive response

A single glycan on IgE is indispensable for initiation of anaphylaxis

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell–bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology

High-resolution physicochemical characterization of different intravenous immunoglobulin products - Fig 4

<p><b>Allotype distribution for the G1m1/nG1m1 isoallotypes by size-exclusion chromatography–time of flight–mass spectrometry (A) and heatmap of positive probes bound by intravenous immunoglobulin (IVIg) products on HuProt™ human proteome arrays (B).</b> A total of 355 proteins on the HuProt™ arrays showed positive binding signals to the nine IVIg samples. These positive probes were clustered in the heatmap according to their A scores reflecting the signal intensities. Red color shows that a given sample has a higher-than-average A score for the given probe, and blue color reflects a lower-than-average A score. Each IVIg sample was probed on 2 arrays labeled as “–#a” and “–#b,” respectively. Single lots of each product were analyzed prior to or after the expiration date: Pri-1 (22 months after), Pri-2 (2 months prior), and Pri-3 (28 months after); Oct-1 (5 months prior), Oct-2 (12 months after), and Oct-3 (24 months after); Gam-1 (6 months after), Gam-2 (36 months prior), and Gam-3 (24 months after). Gam, Gammagard lot; Oct, Octagam lot; Pri, Privigen lot.</p

Size-exclusion chromatography analysis of 3 lots of intravenous immunoglobulin (IVIg) from 3 different manufacturers for a comparison of 9 different IVIg materials.

<p>(A) UV 280 nm detection; peak A, aggregate; peak D, dimer; peak M, monomeric IgG. (B) Abundance of aggregate (peak A, panel A). (C) Abundance of dimer (peak D, panel A). Overlay of intrinsic fluorescence (excitation 280 nm, emission 340 nm; blue) and extrinsic (Bis-ANS) fluorescence (excitation 385 nm, emission 435 nm; black) for (D) Privigen, (E) Octagam, and (F) Gammagard. Overlay of extrinsic fluorescence for fresh (back) and expired (blue) lots of (G) Privigen, (H) Octagam, and (I) Gammagard. Single lots of each product were analyzed prior to or after the expiration date: Pri-1 (22 months after), Pri-2 (2 months prior), and Pri-3 (28 months after); Oct-1 (5 months prior), Oct-2 (12 months after), and Oct-3 (24 months after); Gam-1 (6 months after), Gam-2 (36 months prior), and Gam-3 (24 months after). Gam, Gammagard lot; Oct, Octagam lot; Pri, Privigen lot.</p

Intravenous immunoglobulin product information.

<p>Intravenous immunoglobulin product information.</p

Nonenzymatic glycation analysis of Octagam lots.

<p>(A) Deconvoluted mass spectrum of papain cleaved Fc from Oct-1 (expired 5 months prior to analysis). Highlighted glycoforms labeled with “Hex,” “2Hex,” or “3Hex” indicate modified glycoforms with additional hexose units. (B) Relative quantitation of immunoglobulin G1 Fc glycoforms analysis of papain cleaved Fc from three lots of Octagam showing product age–related glycation; Oct-1 (5 months prior to expiration), Oct-2 (12 months after expiration), and Oct-3 (24 months after expiration). (C) Peptide liquid chromatography–mass spectrometry/mass spectrometry relative quantitation of nonenzymatic glycation of the peptide VSNK*ALPAPIEK from the CH2 domain of immunoglobulin G1. Data in panel C are displayed as mean ± standard deviation (n = 3 technical replicates). *Denotes glycated species. Gam, Gammagard lot; Oct, Octagam lot; Pri, Privigen lot.</p

Mass spectrometry proteomics analysis of non–IgG proteins in intravenous immunoglobulin products.

<p>Data are displayed as mean ± standard deviation (n = 3 technical replicates). Single lots of each product were analyzed prior to or after the expiration date: Pri-1 (22 months after), Pri-2 (2 months prior), and Pri-3 (28 months after); Oct-1 (5 months prior), Oct-2 (12 months after), and Oct-3 (24 months after); Gam-1 (6 months after), Gam-2 (36 months prior), and Gam-3 (24 months after). Gam, Gammagard lot; nPSMs, normalized peptide spectral matches; Oct, Octagam lot; Pri, Privigen lot.</p