Minimal B Cell Extrinsic IgG Glycan Modifications of Pro- and Anti-Inflammatory IgG Preparations in vivo

Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo.


INTRODUCTION
Immunoglobulin G (IgG) antibodies are glycoproteins with pro-and anti-inflammatory effector functions. Thus, binding of IgG to Fcγ receptors (FcγRs) can trigger effector functions such as the release of pro-inflammatory mediators, phagocytosis of opsonized pathogens, or antibody dependent cellular cytotoxicity (ADCC) (1,2). Apart from host defense, IgG autoantibodies play a major role in tissue destruction and inflammation during autoimmune diseases including the primary Sjögren's syndrome, Systemic Lupus Erythematosus or Rheumatoid Arthritis (3). Moreover, pooled IgG preparations from thousands of donors, called IVIg (intravenous immunoglobulin G), are used as an anti-inflammatory treatment in the therapy of several autoimmune diseases, and during chronic inflammation (4).
The sugar moiety attached to each of two conserved asparagine 297 residues in the constant domain 2 (CH2) of the IgG fragment crystallizable (Fc) is essential for both, the pro-and anti-inflammatory activities of IgG (5) and keeps the IgG molecule in its typical horseshoe shape critical for FcγR binding (6)(7)(8). Apart from IgG Fc-glycosylation, about 10-15% of serum IgG contains an N-linked sugar moiety in the IgG fragment antigen binding (Fab) domain, which is generated de novo during the process of IgG hypermutation (9,10). More recent evidence suggests that IgG Fab glycosylation helps to regulate IgG specificity (11). With respect to IVIg, especially terminal sialic acid residues were shown to be responsible for its anti-inflammatory activity. Thus, while desialylated IVIg lost its capacity to suppress a wide variety of autoimmune diseases in mice (12,13), IVIg preparations enriched for terminal sialic acid residues showed an enhanced anti-inflammatory activity (5,14). Of note, enriching cytotoxic antibodies for terminal sialic acid residues decreased their activity in vivo and in vitro in some but not all studies (15). Consistent with this reduced activity a reduction in affinity of highly sialylated IgGs for select activating FcγRs was noted (5). Due to the potent immune modulating functions of select IgG glycoforms, new therapeutic approaches try to alter IgG activity by modulating its glycosylation ex vivo (5,16), or by changing the glycosylation status in the patient by enzymatic approaches (17)(18)(19).
Due to the potent immunomodulatory activity of the IgG sugar moiety, a precise monitoring of therapeutic IgG glycosylation has become standard before using new recombinant antibody preparations or consecutive batches of already approved antibodies in patients. This in-depth characterization relies on the fact that once an IgG antibody is injected into the patient, the sugar structures remain stable and are not subject to in vivo processing. However, recent studies suggest that terminal α2,6-linked sialic acids may be attached independently of the B cell secretory pathway (20,21). According to these results, B cell independent IgG sialylation is achieved in the liver by secreted ST6Gal1 produced by cells lining the liver central veins. As a sugar donor, CMP-sialic acid at least partially derived from degranulating platelets may be used. More recently, it was suggested that in addition to antibodies the surface of cells may also become sialylated through this process (22). With respect to therapeutic antibody preparations, these findings raise the severe concern, that this process may alter the activity of therapeutic antibodies in the patient. Thus, cytotoxic antibodies may become less active due to decreased binding to activating FcγRs, while intravenous IgG preparation may become more active and may in the worst-case lead to an unwanted strong immune suppression. Moreover, the genetic heterogeneity of the human population and age dependent alterations of immune responses may further complicate to predict how stable therapeutic antibodies are in individual patients and with respect to glycosylation.
To address this issue and identify to what extent therapeutic IgG preparations are subject to B cell independent sialylation, we made use of two mouse strains lacking either B cells or ST6Gal1, which is the responsible enzyme for adding terminal sialic acid residues to the IgG sugar moiety (23)(24)(25). Both mouse strains were injected with human IVIg preparations having either a normal or strongly reduced level of sialylated IgG glycoforms. IgG N-glycan analysis by HILIC-UPLC-FLR (plus MS-detection) and xCGE-LIF of mouse serum for several consecutive days after IVIg administration revealed that IVIg glycosylation is very stable upon injection in vivo. Only a very small fraction, predominantly of disialylated IgG N-glycans attached to the Fab-rather than the IgG Fc-portion appeared to increase over time. In summary, our results suggest that B cell extrinsic IgG sialylation may not modulate therapeutic IgG activity post injection in vivo.

ELISA
Sera of 8-9 week old C57BL/6, µMT and ST6Gal1 −/− mice were collected and stored at−20 • C until further use. For quantification of total serum IgM and IgG the Mouse IgM ELISA Quantification Kit and the Mouse IgG ELISA Quantification Kit (Bethyl) were used according to the manufacturer's instructions: ELISA plates were coated with 100 ng/well goat anti-mouse IgM or IgG in Carbonate/Bicarbonate for 1 h at room temperature. After washing unspecific binding was blocked with PBS/1% BSA for 1 h at room temperature. Sera were diluted 1:2,500 (IgM) or 1:10,000 (IgG) in PBS/1% BSA and incubated for 1 h at room temperature and, after adequate washing, bound IgM and IgG antibodies were detected by 1:20,000 diluted (in PBS/1% BSA) anti-IgM-HRP or anti-IgG-HRP antibody in PBS/1% BSA (incubated for 1 h at room temperature). For detection, TMB Solution was added and the reaction was stopped with 6% orthophosphoric acid. OD was measured with VersaMax tunable microplate reader (Molecular Devices) at 450 and 650 nm. For detection of remaining human IVIg or neuraminidase treated IVIg in the sera of ST6Gal1 −/− mice the Human IgG ELISA Quantification Kit (Bethyl) was used according to the above-described protocol. ELISA plates were coated with 100 ng/well goat anti-human IgG. Sera were diluted 1:50,000.

Rituximab-IgG-Induced B Cell Depletion in PBMC Humanized
Rag2/γc/FcεRγ/FcγR2b −/− PBMCs were isolated by density centrifugation from individual buffy coats. Isolated PBMCs were frozen and stored in liquid nitrogen until further use. Adult Rag2/γc/FcεRγ/FcγR2b −/− mice were irradiated with 6 Gy and injected intraperitoneally with 1 × 10 7 human peripheral blood mononuclear cells (PBMCs) 6 h after irradiation as described previously (27). Eighteen hours after PBMC transfer, an equal amount of 0.5 µg anti-CD20 rituximab IgG1 (MabThera R ) in the serum of rituximab injected mice was given intraperitoneally and 24 h later B cell counts in the peritoneum were analyzed by flow cytometry.

IgG Isolation
The IgG was isolated using protein G monolithic plates (BIA Separations, Ajdovščina, Slovenia) as described previously (28).

Methanol Desalting
Volumes of eluates corresponding to 100 µg of IgG were dried in a vacuum concentrator. Samples were then desalted by methanol precipitation. For methanol desalting 1 mL of cold (−20 • C) methanol (MeOH) was added to each sample and resuspended. The plate containing samples was then closed with adhesive seal and centrifuged at 2,200 g for 15 min. After centrifugation, 970 µL of MeOH was discarded and 1 ml of cold MeOH was added again to each sample and resuspended. After that, the plate was again centrifuged at 2,200 g for 15 min. After a second centrifugation, the 970 µL of MeOH again was discarded and the remaining sample was then dried by vacuum centrifugation.

Glycan Release
The dried, desalted samples were dissolved in 30 µL

HILIC-SPE
The samples (in a volume of 75 µL) were mixed with 700 µL of cold 100% ACN (Sigma-Aldrich, St. Louis, MO, USA). Free label and reducing agent were removed from the samples using HILIC-SPE on a 0.2 µm GHP filter plate (Pall Corporation, Ann Arbor, MI, USA). Solvent was removed by application of vacuum using a vacuum manifold (Millipore Corporation, Billerica, MA, USA).
All wells were prewashed using 200 µL of 70% ethanol (Carlo Erba Reagents, Val de Reuil, France), followed by 200 µL water and equilibrated with 200 µL of cold 96% ACN. The samples were loaded onto GHP filter plate and incubated for 2 min before the vacuum application. The wells were subsequently washed 5 × using 200 µL of cold 96% ACN. The last washing step was followed by centrifugation at 165 × g for 5 min. Glycans were eluted two times with 90 µL of ultrapure water after 15 min of shaking at room temperature followed by centrifugation at 165 × g for 5 min. The combined eluates were stored at −20 • C until usage.

HILIC-UPLC-FLR Analysis of IgG Glycans
Fluorescently labeled N-glycans were separated by HILIC on a Waters Acquity UPLC instrument (Milford, MA, USA) consisting of a quaternary solvent manager, sample manager and a fluorescence (FLR) detector set with excitation and emission wavelengths of 250 and 428 nm, respectively. The instrument was under the control of Empower 3 software, build 3471 (Waters, Milford, MA, USA). The UPLC-FLR system was equipped with a hydrophilic interaction liquid chromatography (HILIC) column, a Waters BEH Glycan chromatography column (100 × 2.1 mm i.d., 1.7 µm BEH particles). The separation used a gradient of 75% solvent B (100% ACN; solvent A: 100 mM ammonium formate pH 4.4) to 62% solvent B over 27 min, with a flow of 0.4 ml/min. Solvent B was maintained at 62% for an additional 5 min. The column was then washed for 2 min with 100% of solvent A. Initial conditions were restored in 1 min and held for an additional 5 min to ensure column reequilibration. Samples were maintained at 10 • C before injection, and the separation temperature was 60 • C. The system was calibrated using an external standard of hydrolyzed and 2-AB labeled glucose oligomers from which the retention times for the individual glycans were converted to glucose units (GU). The chromatographic glycan peaks resulting from the HILIC-UPLC-FLR analysis were integrated using an automatic processing method with a traditional integration algorithm after which each chromatogram was manually corrected to maintain the same intervals of integration for all samples. The amount of glycans in each peak was expressed as % of total integrated area. Peak annotation of human and murine IgG was performed according to Pučić et al. (28) and Kristic et al. (29). Sample was maintained at 10 • C before injection, while the separation temperature was 60 • C. Fluorescent detector was set with excitation and emission wavelengths of 250 and 428 nm, respectively. Data processing was performed using an automatic processing method with a traditional integration algorithm after which each chromatogram was manually corrected to maintain the same intervals of integration for all the samples. Relative abundance of each obtained peak was expressed as percentage of total integrated area. Mass spectrometer was operated in a positive ion mode with capillary voltage set to 2,250 V and nebulizing gas at pressure of 5.5 Bar. Drying gas (nitrogen) was applied to source at a flow rate of 4 L/min and temperature of 300 • C, while vaporizer temperature was set to 300 • C and flow rate of 5 L/min. Nitrogen was used as a source gas, while argon was used as collision gas. Ion energy was set to 4 eV, transfer time was 100 µs. Spectra were recorded in m/z range of 50-3,000 at a 0.5 Hz frequency. N-glycan structures were assigned based on retention time, measured mass and fragmentation spectra using GlycoMod (30) (http://web.expasy.org/glycomod/) and GlycoWorkbench (31).

Methanol Desalting
Volumes of IgG eluates corresponding from 3 to 10 µg of IgG were dried in a vacuum concentrator and desalted following previously described protocol for methanol precipitation.

N-Glycan Release
The

Statistical Analysis
The statistical significance of the data was determined as indicated in the figure legends. In brief, the Kruskal-Wallis test, followed by Dunn's multiple comparison test, or repeated measures two-way ANOVA with Bonferroni post-test were used to determine statistical differences between more than two groups. To indicate different levels of significance, a p 0.05 was assigned one asterisk, a value smaller than 0.05 but larger than 0.001 was assigned two asterisks and a value smaller than 0.001 was assigned three asterisks.

Model System to Study B Cell Independent Sialylation of Therapeutic IgG
To determine to what extent therapeutic IgG preparations are subject to B cell independent sialylation, we made use of two mouse strains, namely ST6Gal1 deficient (ST6Gal1 −/− ) and µMT mice. While ST6Gal1 −/− mice lack the sialyltransferase 1, catalyzing the addition of α2,6 linked sialic acid residues (23,35), µMT mice have a disruption of the gene encoding the µ-chain constant region resulting in an arrest of B cell development at the pre-B-cell stage (36). As shown in Figure 1, we started with a characterization of the two in vivo model systems with respect to B cell development and the presence of a2,6-linked sialic acid residues. As expected, µMT mice had no B cells in the blood, while the amount of B cells of ST6Gal1 −/− mice was comparable to C57BL/6 mice ( Figure 1A). Consistent with the absence of B cells in µMT mice, no IgM and IgG antibodies were detectable in the serum ( Figure 1B). In contrast, ST6Gal1 −/− mice showed normal levels of B cells, strongly reduced levels of serum IgM and a trend toward higher levels of serum IgG as described before ( Figure 1B) (35,37). Staining with SNA (sambucus nigra agglutinin)-a plant lectin specifically detecting α2,6 linked sialic acids-demonstrated that ST6Gal1 −/− mice were negative for SNA on B and T cells, whereas µMT mice showed a T cell SNA staining pattern comparable to C57BL/6 mice ( Figure 1C). Finally, the serum IgG glycosylation pattern of C57BL/6, µMT and ST6Gal1 −/− mice was analyzed by HILIC-UPLC-FLR confirming the absence of a2,6 linked sialic acid species on serum IgG of ST6Gal1 −/− mice ( Figure 1D). In addition, we analyzed the sugar structures present in IVIg and neuraminidase digested IVIg preparations (NeuIVIg), which were used for studying B cell independent sialylation in vivo ( Figure 1D). As expected, no sialic acid containing sugar moieties were detectable in neuraminidase digested IVIg, allowing a detection of extrinsic de novo sialylation in vivo with the greatest possible sensitivity.

Analysis of B Cell Extrinsic IVIg Sialylation in vivo
To address if therapeutic IgG preparations are subject to B cell extrinsic de novo sialylation in vivo, we injected 10 mg IVIg in B cell deficient µMT mice and analyzed alterations in IVIg glycosylation in the serum of these animals by hydrophilic interaction ultra-performance liquid chromatography with fluorescence detection (HILIC-UPLC-FLR). In addition, the identity of each peak, were sialylation was expected (GP15-26), was confirmed by mass spectrometry (fragmentation spectra see Figure S1). As mouse IgG glycans terminate with Nglycolylneuraminic acid (Neu5Gc), while human IgGs carry terminal N-acetylneuraminic acids (Neu5Ac), the injection of human IgG into mice allows an unequivocal detection of B cell independent sialylation (29,(38)(39)(40). An overview of all detected murine and human glycan structures is shown in Figure S2. When we compared the HILIC-UPLC-FLR profiles of IVIg before and 6 days after injection in µMT mice we found that the IVIg glycosylation profiles overlapped almost completely (Figure 2A). However, some small additional glycan  peaks (GP20, GP25, GP26) were detected that were not present in the original IVIg preparation (enlarged inset in Figure 2A). Interestingly all of those novel glycan peaks overlapped with sialic acid containing sugar structures (GP20 containing one sialic acid residue, GP25 and GP26 containing two sialic acid residues with (GP26) or without (GP25) fucose) selectively present in serum IgG from C57BL/6 mice (Figure 2B), suggesting that a very small level of extrinsic sialylation of IVIg can occur in mice. We next analyzed the kinetics of B cell independent IgG sialylation by studying changes in IVIg sialylation 2, 4, and 6 days after injection into µMT mice (Figure 3 and Figure S3). As some studies suggest, that the level of IgG sialylation may affect antibody half-life and FcRn binding (41,42), we first assessed if both, sialylated and non-sialylated IVIg preparations had a comparable half-life. As shown in Figure S3A, however, we noted no effect in the half-life of sialylated and asialylated IVIg (5), ensuring that both IVIg preparations had a comparable chance to become resialylated in vivo. Indeed, the presence of both disialylated sugar structures (GP25 and 26) slowly increased over time (Figure 3A). To increase the amount of acceptor sites for extrinsic IgG sialylation we also injected IVIg pretreated with neuraminidase (NeuIVIg). As shown in Figure 3B, however, this only mildly increased the level of extrinsic IgG sialylation on afucosylated sugar moieties, despite the availability of large amounts of IgG-G2 glycosylation variants. In total, only 1% of IgG glycovariants present in NeuIVIg acquired a disialylated sugar moiety 6 days after injection into µMT mice. To further validate these results, we repeated this experiment in ST6Gal1 −/− mice, which are not able to add α2,6linked sialic acid residues and therefore served as a negative control. As shown in Figures 3C,D and Figure S3, the sialylation of IVIg or neuraminidase digested IVIg did not change over time in these animals, suggesting that the increase in GP25 and GP26 were indeed due to de novo IgG sialylation by ST6Gal1. In contrast, the small increase in monosialylated fucosylated IgG glycostructures (GP20) was also evident in ST6Gal1 deficient mice (Figures S3B,C). Moreover, afucosylated monosialylated glycan forms (GP18, A2G2Z1) of IVIg or NeuIVIg ( Figure 4B) did not increase over time ( Figure S3). Importantly however, the monosialylated glycoforms in GP18 comprise almost completely of human FA2G2S1 and it is therefore difficult to quantify murine monosialylated glycostructure by HILIC-UPLC-FLR. This prompted us to perform further studies.

Detection of B Cell Extrinsic IgG Sialylation by xCGE-LIF
To ensure that the inability to detect changes in de novo generated monosialylated IgG glycoforms was not due to technical reasons, we decided to use multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) to confirm our results. The advantage of xCGE-LIF is that very small amounts of IgG preparations can be analyzed with high sensitivity allowing better resolution for sialylated species. The characteristic glycan profiles of murine and human IgG preparations by xCGE-LIF as well as the structure and peak assignment are depicted in Figure S4. Based on the fact that neuraminidase digested IVIg allowed the most clear-cut identification of de novo IgG sialylation in vivo, we focused on NeuIVIg-injected µMT and ST6Gal1 −/− mice (Figures 4, 5). As shown for HILIC-UPLC-FLR analysis, additional glycan peaks became detectable on NeuIVIg 6 days after injection in B cell deficient µMT mice, which overlapped with IgG sugar structures selectively present on mouse but not human IgG (Figure 4B). Fully consistent with HILIC-UPLC-FLR, the glycan peaks that appeared de novo and were increasing over time were mono-(P11 and P13) and disialylated sugar structures (P2 and P4) with (P4 and P13) or without (P2 and P11) core-fucose (Figures 4, 5). Injection of NeuIVIg into ST6Gal1 −/− mice did not lead to any detectable changes in IVIg sialylation in vivo (Figures 5B,C). Moreover, agalactosylated (G0), mono-galactosylated (G1), and digalactosylated (G2) IgG glycoforms were not changed over time ( Figure S5). Thus, xCGE-LIF analysis allowed a better detection of mono-and disialylated IgG sugar structures.
To exclude that the observed minimal level of extrinsic IgG sialylation is due to the use of human IgG in mice, we performed a similar experiment where we injected 1 mg of the murine TA99-IgG2c antibody-directed against the glycoprotein 75into B cell deficient µMT mice and analyzed alterations in IgG glycosylation in the serum of these animals 10 min as well as 2, 4, and 6 days after injection by xCGE-LIF. As shown in Figure S6 murine IgG acquired sialic acid residues to a similar extend as human IgG. Again, especially disialylated sugar structures (P2 and P4) were increasing over time. The sugar structure FA2G2Z1 (P13) might co-migrate with the large peak present in initial TA99-mIgG2c antibody (around 250 MTU") and therefore could not be clearly distinguished. In summary, the data obtained with HILIC-UPLC-FLR and xCGE-LIF analysis suggest that a very small amount of IgG molecules-both human and murinecontaining two galactose residues can become modified with one or two sialic acid residues in vivo in the absence of B cells.
To further characterize extrinsic IgG sialylation, we also discriminated de novo generated IgG glycoforms between Fab and Fc. For this purpose, the isolated serum IgG preparations from NeuIVIg injected µMT mice (4 days after NeuIVIg injection) were separated into Fab and Fc portions before glycoanalysis by xCGE-LIF. Individual analysis of the separated IgG fragments revealed that extrinsic IgG sialylation occurred almost exclusively on N-glycosylation sites of the Fab (Figure 6A) but not of the Fc (Figure 6B) fragment. Again, minimal amounts of especially disialylated IgG sugar structures were appearing. In the two mouse serum samples 1.57 ± 0.3% of all IgG glycoforms on the Fab fragment corresponded to disialylated sugar structures, while only 0.89 ± 0.15% were monosialylated. In contrast, no additional murine sialic acids could be detected on the Fc fragment ( Figure 6B). Therefore, predominantly the Nlinked sugar moieties of the easily accessible Fab fragment in the IVIg preparations seems to be the target for de novo sialylation.

Impact of Extrinsic Sialylation on IgG Effector Function
To evaluate the impact of extrinsic sialylation on cytotoxic IgG effector functions, we injected µMT and ST6Gal1 −/− mice with the CD20 specific antibody rituximab, which is broadly used in patients with autoimmune diseases and cancer. As rituximab selectively recognizes human but not mouse CD20, the injection into ST6Gal1 deficient mice with Rituximab does  not affect B cell numbers. Four days after injection, serum from µMT and ST6Gal1 knockout mice was collected, the level of human IgG determined and extrinsic IgG sialylation assessed by xCGE-LIF. As shown in Figure 7A, very low amounts of exclusively disialylated IgG structures were found in rituximab injected µMT mice. To assess the functional activity of human CD20 antibodies present in the serum of µMT and ST6Gal1 deficient mice, a serum equivalent containing 0.5 µg rituximab was injected into immunodeficient Rag2/γc/FcεRγ/FcγR2b −/− mice, which were irradiated and reconstituted with human peripheral blood mononuclear cells 18 h before ( Figure 7B). As shown in Figures 7C,D, rituximab treatment via serum transfer of rituximab-injected µMT and ST6Gal1 −/− mice led to a significant depletion of B cells in the peritoneal cavity, while B cell counts in PBS-serum treated mice were not affected. In line with the low efficiency of the extrinsic sialylation pathway, when we compared B cell depletion between rituximab-containing ST6Gal1 −/− and µMT serum, no significant differences in cytotoxic antibody activity were detected.

DISCUSSION
An important step in the process of introducing a new therapeutic IgG antibody or a new batch of an already approved antibody into the clinic is an in-depth characterization of the IgG glycovariants present in the antibody preparation. It is well-known that many factors, such as the cell line in which a therapeutic antibody is produced (43) or the specific culture conditions (44), can alter IgG glycosylation. The importance of detecting alterations in IgG glycosylation have been emphasized by the fact that relatively minor changes in the composition of the biantennary complex sugar structure attached to both of the IgG Fc-domains can dramatically alter IgG activity. Thus, IgG antibodies lacking fucose, sialic acid, and galactose have been described to have an enhanced pro-inflammatory activity, while antibody glycoforms rich in terminal sialic acid residues have an active anti-inflammatory and immunomodulatory activity (5,14,45,46). Until recently, it was believed that IgG glycosylation is established exclusively within the cells in which the antibody is produced and remains rather stable upon injection in vivo. However, more recent evidence suggests that IgG glycosylation may be actively altered in vivo (20,21,47). Thus, IgG antibodies were described to become sialylated post secretion from plasma cells. As enhanced IgG sialylation was shown to impact IgG activity, this would represent a major concern for the use of therapeutic antibodies in patients, prompting us to analyze to what extent this extrinsic IgG sialylation pathway affects passively transferred antibodies. Technically this represents a major challenge as in vivo several factors may lead to an altered abundance in IgG glycoforms over time. For example, a more rapid clearance of select IgG isotypes or glycoforms may lead to an increase in other IgG glycoforms with a longer IgG half-life (41,48). In a similar manner, using mouse strains with a cell subset specific deletion of ST6Gal1, the enzyme that catalyzes the addition of α2,6-linked terminal sialic acid residues on IgG antibodies, may overestimate the level of B cell extrinsic sialylation if the deletion in the target cell population is incomplete and if B cell subsets that have escaped ST6Gal1 FIGURE 7 | Impact of IgG de novo sialylation on cytotoxic antibody activity. Shown is a representative overlay of xCGE-LIF IgG glycoanalysis of rituximab (RTX) treated µMT mice (A), a schematic overview of the experimental setup (B), and its respective results (C,D). (A) µMT mice were injected with 400 µg RTX (µMT RTX) and 4 days later serum IgG was analyzed by xCGE-LIF to identify de novo generated IgG glycoforms. Schematic drawings of the sugar moieties are depicted for the major peaks. Enlarged insets emphasize peaks detected selectively on RTX 4 days post injection into µMT mice. (B) Immunodeficient Rag2/γc/FcεRγ/FcγR2b −/− mice were irradiated (6 Gy) and reconstituted with human PBMC in the peritoneal cavity. Eighteen hours later mice (n = 5-6) received equal amounts of rituximab (0.5 µg/20 g mouse) derived from µMT or ST6Gal1 −/− mice, which had been injected with rituximab 4 days before. Twenty-four hours after RTX injection, cells of the peritoneal cavity were analyzed by flow cytometry. Shown are representative FACS plots (C) and the quantification (D) of human B cell counts in the peritoneal cavity 1 day after rituximab or PBS injection. Statistical significance was evaluated with a Kruskal-Wallis test followed by Dunns post-hoc test. **p = 0.01, ***p = 0.001. Horizontal lines represent median values.
deletion have a competitive advantage over those with a deletion of the enzyme.
To study this in a most informative setting we decided to analyze changes in human IgG sialylation in mice lacking mature B cells and serum antibodies. To get the most complete picture we used human intravenous immunoglobin preparations (IVIg), as IVIg contains all human IgG subclasses present in serum. In this experimental scenario, human IgG molecules represent the only antibody isotype in the serum and hence should be fully accessible for de novo sialylation. Moreover, mouse terminal sialic acid residues (Neu5Gc) can be distinguished from human sialic acid residues (Neu5Ac) by analytical techniques allowing an unequivocal identification of newly added mouse derived sialic acid residues to the transferred human antibodies in vivo (38). By using two independent analytical techniques to assess extrinsic de novo IgG sialylation in vivo, our results suggest that the addition of sialic acid residues is a very rare, but nonetheless detectable event. Interestingly the acceptor sugar moieties accessible for the de novo mono-or disialylation seemed to contain two terminal galactose residues (G2 glycoform), while in principle also monogalactosylated (G1) glycoforms could have served as acceptor structures for de novo sialylation and are normally present in mice in vivo. Moreover, both core fucosylated G2 and non-fucosylated G2 sugar moieties were able to acquire terminal Neu5Gc. Of note, increasing the amount of potential acceptor sugar structures by pre-treating human IgG with neuraminidase only marginally increased the level of IgG sialylation, further strengthening the notion, that extrinsic IgG sialylation is a rather inefficient process. However, it has to be considered that also the monosialylated structures as potential acceptor structures were removed. More importantly, when IgG preparations such as IVIg were used, in which IgG molecules present additionally containing N-linked sugar moieties in the Fab portion as potential acceptor sites for extrinsic sialylation, an almost exclusive sialylation occurred in these more easily accessible sugar domains (although not at a higher efficacy). As Fab arm glycosylation may modify antibody specificity, it will be interesting to study if extrinsic Fab arm glycosylation affects target antigen binding (49). If no Fab associated sugar moieties were present, however, minor changes in Fc-linked sugar moieties could be detected for mouse and human monoclonal antibodies. As the extent of B cell extrinsic IgG sialylation was similar for mouse and human IgG, we would exclude that human IgG molecules become sialylated less efficiently in mice due to some species barrier effects. Furthermore, as the amount of de novo sialylated IgG never exceeded two percent of the total IgG present in vivo, one would not expect functional consequences for IgG activity. Indeed, the human CD20 specific antibody Rituximab did not show an altered activity when passaged through B cell deficient mice.
In summary, our study demonstrates that the process of B cell extrinsic de novo sialylation of IgG antibodies in vivo is only affecting a minor subset in the pool of IgG glycovariants present in IVIg and cytotoxic IgG preparations and hence may not trigger altered pro-or anti-inflammatory IgG activities.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the government of Lower Franconia.