Fcγ RIIIa chromatography to enrich a-fucosylated glycoforms and assess the potency of glycoengineered therapeutic antibodies

Therapeutic antibodies can elicit an immune response through different mechanisms, either cell independent via complement activation (CDC) or through activation of immune-effector cells (such as macrophages and NK cells).

Journal of Chromatography A 1610 (2020) 460554 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Fcγ RIIIa chromatography to enrich a-fucosylated glycoforms and assess the potency of glycoengineered therapeutic antibodies Anne Freimoser–Grundschober a,∗, Petra Rueger b, Felix Fingas b,1, Peter Sondermann a,2, Sylvia Herter a, Tilman Schlothauer b, Pablo Umana a, Christiane Neumann a a b Roche Pharma Research & Early Development, Roche Innovation Center Zurich, Roche Glycart AG, Wagistrasse 10, CH-8952 Schlieren, Switzerland Roche Pharma Research & Early Development, Roche Innovation Center Munich, Nonnenwald 2, 82377 Penzberg, Germany a r t i c l e i n f o Article history: Received 20 June 2019 Revised 12 September 2019 Accepted 17 September 2019 Available online 18 September 2019 Keywords: ADCC Affinity column Fcγ RIIIa Fucose Glycoengineering Monoclonal antibody a b s t r a c t Therapeutic antibodies can elicit an immune response through different mechanisms, either cell independent via complement activation (CDC) or through activation of immune-effector cells (such as macrophages and NK cells) After target binding, the Fc part of the antibody will interact with Fc receptors on the surface of effector cells, leading to activation and lysis of the target cells by a mechanism called antibody-dependent cell-mediated cytotoxicity (ADCC) The ADCC of an antibody can be increased by modifying the carbohydrates on the Fc part If the fucose on the first N-acetylglucosamine is absent, the affinity for the Fcγ RIIIa is increased and the ADCC enhanced We describe the development of a chromatography method that is based on the differential affinity of the Fc receptor Fcγ RIIIa (high affinity V158 variant) for fucosylated and a-fucosylated antibodies Immobilized Fcγ RIIIa can be used for the separation of immunoglobulins carrying these glycosylation variants for both, analytical and preparative purposes The biological activity and fucose content of three pools enriched for fully fucosylated, monofucosylated or a-fucosylated carbohydrates could be characterized Mono-fucosylated and a-fucosylated immunoglobulins have the same enhanced biological activity compared to fully fucosylated IgGs A direct, label- and modification-free analytical method for screening of IgGs from culture supernatant was developed and was amenable to high-throughput screening Clones producing antibodies with a high content of a-fucosylated oligosaccharides could be successfully selected © 2019 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction The number of antibody-based therapeutics, either approved or in clinical trials, is increasing constantly and the majority is based on the human IgG1 isotype [1,2] The two Fab (fragment antigen binding)-parts of these IgGs are responsible for target binding, whereas the constant Fc (fragment crystallizable) domain interacts with components of the immune system, leading to mediation of immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) The carbohydrate structures attached to the conserved N-glycosylation site at Asparagine 297 (Asn297, N297) within the ∗ Corresponding author E-mail address: anne.freimoser-grundschober@roche.com (A Freimoser– Grundschober) Present Address: GVG Diagnostics, GmbH, Deutscher Platz 5b, 04103 Leipzig, Germany Present Address: Tacalyx GmbH, Müllerstr 178, 13353 Berlin, Germany CH2-domain of the constant Fc part are mandatory for mediating these effector functions These oligosaccharides consist predominantly of a bi-antennary core pentasaccharide, comprised of Nacetylglucosamine and mannose, and complex type structures with a variable content of bisecting GlcNAc (N-acetyl-glucosamine), terminal galactoses, core fucose and sialic acids (Fig 1) In addition, these oligosaccharides may be differently composed on each of the two heavy chains of the same IgG molecule Numerous studies have shown that the carbohydrates play an important role in maintaining the structure and stability of the IgGs and that the carbohydrate composition strongly affects the antibody-mediated immune effector functions Indeed, the Fcattached oligosaccharides influence the affinity of the antibody for individual Fcγ Rs and regulate binding to different Fcγ R classes: activating or inhibitory [1,3–8] To improve the biological functions of therapeutic antibodies, several approaches have been developed to modulate their glycosylation profile [2–4,9–11] A promising approach is the reduction or abolishment of the core fucosylation, because the absence of the core fucose results in a 50-fold https://doi.org/10.1016/j.chroma.2019.460554 0021-9673/© 2019 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) A Freimoser–Grundschober, P Rueger and F Fingas et al / Journal of Chromatography A 1610 (2020) 460554 Fig Carbohydrate moiety attached to Asn-297 of human IgG1-Fc The sugars in bold define the pentasaccharide core; the addition of the other sugar residues is variable GlcNAc, N-acetylglucosamine; Fuc, fucose; Man, mannose; Gal, galactose; NeuAc, N-acetylneuraminic acid increased antibody affinity to Fcγ RIIIa and Fcγ RIIIb, leading to enhanced ADCC activity [12,13] At least three antibodies in clinical trials have carbohydrates with reduced fucosylation on their Fc part: obinutuzumab [14], MEDI-551 [15] and GSK2831781 (ClinicalTrials.gov identifier: NCT02195349) During clone screening of a stable production cell line for a classical monoclonal antibody, the major selection criterion is the titer of the secreted antibody (usually determined by ProteinA binding) In the case of antibodies with a-fucosylated carbohydrates, it is also critical to monitor the oligosaccharide composition in addition to the titer The carbohydrate analysis can be performed with different methods that usually involve digestion of the oligosaccharides from the IgGs and analysis via reverse-phase UPLC with previous labeling of the carbohydrates or without labeling by mass spectrometry These methods are tedious and only partially adaptable to high-throughput analysis The conventional affinity chromatography matrices applied for IgG purification cannot discriminate between different glycosylation patterns within the IgG, since the immobilized capture proteins specifically bind the protein backbone of an antibody For example, protein A and protein G bind to the interface between the CH2 and CH3 domain of the Fc-part, whereas protein L interacts with the constant part of the kappa light chain A recently published report using an immobilized non-glycosylated Fcγ RIIIa could not differentiate between glycoforms that were fucosylated or not [16] Another approach takes advantages of carbohydrate binding lectins Lectin chromatography, applied to enrich antibodies carrying defined carbohydrates [17], as well as a lectin ELISA developed in house (data not shown), are, however, limited by their inability to bind glycoproteins lacking a specific monosaccharide unit, which is the case for a-fucosylated antibodies Since the higher Fcγ RIIIa affinity of a-fucosylated antibodies results in an enhanced ADCC, screening for Fcγ RIIIa binding has the potential to identify antibodies with improved biological activity Here, we describe a chromatography method that is based on the differential affinity of the Fc receptor Fcγ RIIIa (high affinity V158 variant) for fucosylated and non-fucosylated antibodies Immobilized Fcγ RIIIa can be used for the separation of immunoglobulins carrying these glycosylation variants for both, analytical and preparative purposes Preparative Fcγ RIIIachromatography enables the enrichment of non-fucosylated or fucosylated immunoglobulins for the detailed characterization of their biological activity Analytical Fcγ RIIIa-chromatography can be employed for characterizing the carbohydrate composition of an antibody pool, as well as for the high-throughput screening of clones producing antibodies with a high content of nonfucosylated oligosaccharides, the read-out correlating directly to cell-mediated killing (e.g enhanced ADCC) Results 2.1 Preparative separation of IgG with different a-fucosylation degree An antibody pool comprises antibodies with different degrees of fucosylation (i.e fully fucosylated antibody species, when both carbohydrates of the Fc carry a fucose residue, mono- and afucosylated species if only one or no fucose is present, respectively) For the preparative separation of IgGs with different fucose content, a column with a volume of 2.7 ml and with the Fcγ RIIIa(V158)K6H6 receptor coupled to NHS-sepharose beads was used to isolate these different species from such an antibody pool The IgGs were loaded at pH 8.0 and eluted in three steps at pH 4.6, pH 4.2 and pH First, the GEmAb1 (glycoengineered monoclonal antibody 1) was subjected to preparative Fcγ RIIIa chromatography analysis The chromatogram showed three peaks corresponding to the flow through, and the elution steps with pH 4.6 and pH 4.2 No additional material eluted in the last wash at pH The respective fractions of the peaks were collected for further analysis of their carbohydrate content, their binding to the Fcγ RIIIa by surface plasmon resonance (SPR) and their ability to induce ADCC The same procedure was subsequently performed for GEmAb2 (Fig 2) 2.1.1 Analysis of the carbohydrate composition The carbohydrate composition of the antibody samples separated by Fcγ RIIIa affinity column was analyzed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) following a PNGaseF treatment to release the carbohydrates from the IgG For the two glycoengineered IgGs that were analyzed here, the flow through peak had the lowest afucose content, followed by peaks two and three (Tables and 2) However, this approach delivered only the overall amount of nonfucosylated oligosaccharides in the antibody preparation To determine the distribution of fucose residues on the two antibody heavy chains in the Fc domain, the samples were digested either with plasmin, EndoH and EndoS (GEmAb1), or with EndoH and EndoS (GEmAb2), to obtain Fc fragments (GEmAb1) or whole IgGs (GEmAb2) carrying only the first N-acetylglucosamine residue (with or without fucose) of the oligosaccharide core (both procedures allow equivalent quantification of fucose distribution per Fc) These Fc fragments or IgGs were subsequently subjected to electrospray ionization mass spectrometry (ESI-MS) analysis to determine the distribution of the fucose per Fc fragment (Tables and 2) The ESI-MS analysis of antibodies and revealed 98% and 88% fucosylated antibodies, respectively, in the flow through fraction (peak 1) The fraction obtained by elution with pH 4.6 (peak 2) consisted of a mixture of all fucosylation types, but antibodies with mono-fucosylated oligosaccharides prevailed (64% and 68.5%, respectively) The fraction of GEmAb1 eluted with pH 4.2 (peak 3) consisted of 61% a-fucosylated and 39% mono-fucosylated IgGs The corresponding fraction of GEmAb2 demonstrated a higher proportion of antibodies with a-fucosylated carbohydrates (76.5%) and the mono-fucosylated and fucosylated species were represented by 18.5% and 5%, respectively In summary, the results demonstrate that the preparative Fcγ RIIIa column can be successfully employed to enrich a-fucosylated antibodies For both, GEmAb1 and GEmAb2, the first peak was strongly enriched in antibodies with fucosylated sugars on both Fc parts, the peak two comprised mostly Fcs with one fucosylated and one a-fucosylated carbohydrate, and the A Freimoser–Grundschober, P Rueger and F Fingas et al / Journal of Chromatography A 1610 (2020) 460554 Fig Preparative Fcγ RIIIa chromatography Chromatogram A280 for GEmAb2 GEmAb2 elutes in three peaks: peak one is the flow-through of the column, peak and elute with two pH steps (pH 4.6 and pH 4.2) Fractions pooled for peak 1, and are indicated Black solid line: A280, black dashed line: pH gradient, grey solid line: pH-value Table Content of carbohydrates with and without fucose for antibody pools of GEmAb1 separated in three peaks by Fcγ RIIIa chromatography A-fucosylation level was determined globally by MALDI TOF MS after PNGase F treatment (average from runs) or the fucose distribution per Fc was determined by ESI-MS after Plasmin/Endo S/Endo H digest (pool of runs) Fractions overall % a-fuc (MALDI) ± std dev GEmAb1 peak GEmAb1 peak GEmAb1 peak GEmAb1 3.9 ± 0.5% 66.7 ± 1.5% 91.9 ± 1.1% 58.5 ± 1.3% % of Fc without fucose with one fucose with two fucoses 1% 22% 61% 30% 1% 64% 39% 41% 98% 14% 0% 29% Table Content of carbohydrates with and without fucose for antibody pools of GEmAb2 separated in three peaks by Fcγ RIIIa chromatography A-fucosylation level was determined globally by MALDI-TOF MS after PNGase F treatment (average from runs) or the fucose distribution per IgG was determined by ESI-MS after Endo S/Endo H digest (pool from runs) Fractions overall% a-fuc (MALDI) ± std dev GEmAb2 peak GEmAb2 peak GEmAb2 peak GEmAb2 ± 4% 65 ± 0.7% 97 ± 0.4% 72% % of Fc without fucose with one fucose with two fucoses 4.5% 20.5% 76.5% 43% 4.5% 68.5% 18.5% 40% 88% 11% 5% 17% population of peak three contained predominantly completely afucosylated antibodies 2.1.2 Surface plasmon resonance The three antibody pools isolated from the Fcγ RIIIa column were expected to possess different affinities for the receptor, since they eluted more or less easily These affinities were determined by SPR for GEmAb2, because the recombinant antigen was available The antibody samples were captured to the antigen immobilized on the chip surface and soluble Fcγ RIIIa was injected The affinities fell in two categories: the fucosylated fraction (peak 1) as well as the WTmAb2 (not glycoengineered, carrying wild-type fucosylated carbohydrates) had KDs around 70 nM, with rapid on and off rates, whereas the peak two and peak three fractions, as well as the glycoengineered GEmAb2, had KDs of around nM and a much slower off rate (Table 3) The IgGs of the peak three, with the highest proportion of antibodies with a-fucosylated carbohydrates, had the highest Fcγ RIIIa binding affinity SPR thus confirmed the correlation between elution time, affinity to Fcγ RIIIa and a-fucosylation level 2.1.3 Antibody-dependent cell-mediated cytotoxicity To assess if the measured affinities correlate with biological activity, the ADCC activity of the separated antibody species of both test IgGs were examined in a cellular assay (as described in the experimental section) The analysis of the ADCC activity revealed A Freimoser–Grundschober, P Rueger and F Fingas et al / Journal of Chromatography A 1610 (2020) 460554 Table Affinity between Fcγ RIIIa and pools of GEmAb2 separated in three peaks by Fcγ RIIIa chromatography KD obtained by surface plasmon resonance at 25 °C The three peaks of the antibody pool of GEmAb2 separated by Fcγ RIIIa chromatography, the starting material and the WTmAb2 with wild type carbohydrates were captured on immobilized antigen and the Fcγ RIIIa was used as analyte Values represent the average ± standard deviation of SPR measures from the fractions of two independent Fcγ RIIIa chromatography runs (values without standard deviation are single measure) Fitting: kinetic (1:1 binding) or steady state (for interactions with too fast on and off-rates) Percentage of overall a-fucosylation determined by MALDITOF MS GEmAb2 peak GEmAb2 peak GEmAb2 peak GEmAb2 start (glycoengineered) WTmAb2 (not glycoengineered) Overall a-fucosylation level (MALDI) KD [nM] Model ± 4% 65 ± 0.7% 97 ± 0.4% 72% 8% 68 ± 13 ± 0.2 1.9 ± 0.1 2.7 ± 0.4 71 Steady state Kinetic Kinetic Kinetic Steady state Fig Biological activity of antibody fractions collected from preparative Fcγ RIIIa chromatography: ADCC assays were performed for the three peaks and the starting material for GEmAb1 (A) and GEmAb2 (B) Black squares: starting material, white diamonds: peak one, black triangles: peak two, black circles: peak three, white squares (only shown in B): WTmAb2 wild-type (not glycoengineered) again a separation in two categories: the antibodies containing mainly fucosylated carbohydrates on both heavy chains (peak one and wild-type, not glycoengineered IgG) had a lower ability to induce ADCC than antibody species from elution peaks two and three, as well as glycoengineered IgG, which demonstrated a similar and higher ability to induce ADCC (Fig 3) For instance, GEmAb1 peak two fraction contained ∼60% mono-fucosylated antibodies, whereas peak three had the same amount of completely a-fucosylated antibodies, but the ADCC assay demonstrated almost identical results These cell-based activity measurements thus confirmed and agreed with the KD determined by SPR Taken together, these results demonstrated that only one afucosylated glycan per Fc was enough for a maximum affinity to Fcγ RIIIa and a corresponding ADCC activity of the antibody Therefore, an overall a-fucosylation level of 50% is enough to obtain the benefits of glycoengineering (i.e increased affinity for Fcγ RIIIa and increased ADCC) Based on this finding, we developed a high- throughput screening method that allowed separating IgGs with double-fucosylated carbohydrates from IgGs with mono- and afucosylated carbohydrates and quantifying of the respective fractions 2.2 High-throughput screening for selection of clones with highly a-fucosylated carbohydrates To identify clones producing a-fucosylated carbohydrates during cell line generation for glycoengineered antibodies, a highthroughput screening method was established For this method, a small Fcγ RIIIa column (60 μl volume) with Fcγ RIIIa coupled to POROSTM material via amine coupling was used and allowed a short run time of per sample The IgGs were loaded at pH 8.0 and eluted in a step at pH 3.0 First, wild type mAb1 and mAb2, as well as their respective glycoengineered variants (carrying high proportions of a-fucosylated A Freimoser–Grundschober, P Rueger and F Fingas et al / Journal of Chromatography A 1610 (2020) 460554 Fc-oligosaccharides) were analyzed The chromatograms, obtained by monitoring absorption at 280 nm, showed two peaks: the flowthrough peak and the elution peak Because the area of the second peak quantifies the antibody fraction with enhanced ADCC activity as confirmed by respective analyses, we calculated the relative biological activity (RBA) of an antibody as the percentage of the second peak area over the total area The RBA of glycoengineered GEmAb1 and was determined to be 66% and 75%, respectively The a-fucose content obtained by MALDI analysis for these antibodies was 48% and 75% respectively For the wild type antibodies WTmAb1 and 2, the RBA was of 26% and 31%, whereas the a-fucose content determined by MALDI was 10% and 9%, respectively This test supported the use of Fcγ RIIIa affinity chromatography to identify antibodies highly enriched in a-fucosylated carbohydrates Next, wild-type antibodies mAb1 or mAb2 and their respective glycoengineered variants were mixed in different proportions to obtain samples with different fucosylation levels These samples were subsequently analysed by Fcγ RIIIa chromatography and MALDI TOF MS The relationship between the percentage of afucosylation assessed by MS analysis and the RBA measured by Fcγ RIIIa chromatography column was linear Five different mixtures were assessed in triplicates and a linear regression was fitted through the obtained 15 data points For mAb1 the slope was 0.939 and the intercept 18.35 with a standard error of 0.024 for the slope and of 0.772 for the intercept The correlation coefficient R2 was 0.991 For mAb2 the slope was 0.645 and the intercept 26.80 with a standard error of 0.007 for the slope and of 0.343 for the intercept The correlation coefficient R2 was 0.998 These results demonstrated that the fucosylation level determined by Fcγ RIIIa affinity chromatography correlates to the actual level of fucosylation measured by MS analysis Next, we tested this screening method on 53 culture supernatants from a clone screen instead of using purified antibodies Cell culture supernatants of different clones expressing the glycoengineered GEmAb3 were analyzed in parallel by two different methods: (1) Protein A chromatography followed by MALDI TOF MS of the released carbohydrates and (2) Protein A chromatography with subsequent Fcγ RIIIa chromatography For this highthroughput analysis, 100 μl of supernatant were injected on a Protein A chromatography column The eluate was subsequently neutralized and either digested with PNGase F for MALDI TOF MS analysis of the carbohydrates or injected onto the Fcγ RIIIa affinity column For the latter approach, the injected sample volume was adjusted, to contain 10 μg of the analyte antibody The Protein A purified samples were eluted into a 96-well plate, which could directly be used for subsequent Fcγ RIIIa chromatography without any additional buffer exchange or pipetting step The RBA (which corresponds to the a-fucose level of the sample) was compared to the percentage of a-fucosylation determined by MALDI TOF MS A similar ranking was obtained with both methods (Fig 4), demonstrating that Fcγ RIIIa affinity chromatography, combined with Protein A purification, is a powerful tool for the high throughput screening of cell culture supernatants and for the ranking of clones according to their titer and a-fucosylation grade 2.3 Fcγ RIIIa affinity chromatography with Fc-dimerized Fcγ RIIIaV158 Soluble, his-tagged Fcγ RIIIa is difficult to produce, results in low yields after purification (up to 14 mg/L), and needs to be chemically coupled to the chromatographic material, which might lead to decreased receptor binding To circumvent these problems, a new construct was designed The C-terminus of the ex- Fig Comparison of Protein A chromatography followed by MALDI TOF MS and Protein A chromatography with subsequent Fcγ RIIIa chromatography as two different methods to analyze the a-fucosylation degree of antibodies purified from cell culture supernatant: Similar ranking obtained by RBA from Fcγ RIIIa chromatography as by MALDI TOF MS of glycoengineered GEmAb3 53 clones of GEmAb3 were analyzed tracellular domain of Fcγ RIIIa was fused to an AviTag for sitespecific biotinylation, followed by an IgA protease cleavage site and the Fc region of an IgG1, which included the P329G, L234A and L235A amino acid substitutions to avoid interactions of the Fc part with the Fcγ RIIIa [18] The Fcγ RIIIa-Avi-Fc PG LALA was expressed and purified as described in material and methods section and the resulting expression yields were increased by a factor of 5.5 (up to 78 mg/L) compared to the construct without the Fc fusion The engineered Fcγ RIIIa-Fc-fusion protein was subsequently biotinylated via the AviTag, and mg were coupled to Streptavidin Sepharose and packed into a ml XK column Fifty micrograms IgG were loaded at pH 6.0 and eluted with a gradient to pH 3.0 Upon optimization of the elution gradient, it was possible to shorten the time of analysis to 10 while retaining resolution Chromatography with this column also resulted in two peaks, as with the Fcγ RIIIa(V158)K6H6-tagged construct (Fig 5): the first one containing the fully fucosylated IgG species and the second the mono- and a-fucosylated IgGs In contrast to the his-tagged construct, the first peak was not the flow-through, but eluted within the pH gradient The binding of the IgG to the immobilized, enzymatically biotinylated Fcγ RIIIa-Fc fusion was stronger than for the chemically coupled his-tagged receptor Nevertheless, we observed also here a linear relationship between the RBA and the fucosylation level determined by reversed phase (RP)-UPLC as observed for the Fcγ RIIIa his construct Five mixtures of GEmAb4 and WTmAb4 were analyzed on this column and compared to the a-fucosylation level determined by RP-UPLC The slope was 0.901 and the intercept -5.11 The correlation coefficient R2 was 0.995 For comparison the Fcγ RIIIa-Avi-Fc PG LALA construct was treated with IgA protease to remove the Fc-polypeptide, biotinylated and coupled to SA-sepharose before packing into a column Comparative analysis of the retention profiles obtained by the two affinity columns demonstrated similar chromatography performance in separating antibody samples by their fucose content, confirming that the Fc fusion had no influence on the chromatography result (data not shown) Finally, we examined the stability and longevity of the Fcγ RIIIaFc affinity column The chromatography results were highly reproducible for up to 500 runs and no loss of receptor activity could be detected (Table 4) 6 A Freimoser–Grundschober, P Rueger and F Fingas et al / Journal of Chromatography A 1610 (2020) 460554 Fig Elution profile of two differently fucosylated antibodies from cell line development obtained by analytical Fcγ RIIIa-Avi-Fc chromatography Peak1 fully fucosylated IgG species, peak2 at least one arm a-fucosylated; black dashed line: medium a-fucosylation level, black solid line: high a-fucosylation level Table Stability of the Fcγ RIIIa chromatography column The same probe (50 μg of WTmAb5) was injected 500 times on the same column The total peak area as well as the percentage of peak and peak were recorded The area and percentages remained stable over 500 runs Run number Total area (mAU) Area peak [%] Area peak [%] run run run run run run 134 138 140 135 138 140 83.5 83.4 83.3 83.5 83.3 83.3 16.5 16.6 16.7 16.5 16.7 16.7 100 200 300 400 500 Discussion High levels of a-fucosylated oligosaccharides result in strong Fcγ RIIIa binding and increased biological activity in ADCC assay for glycoengineered therapeutic antibodies [6,19] In this study, we present a Fcγ RIIIa based chromatography to separate antibodies according to the degree of fucosylation of their Fc-attached carbohydrates The method presented here allowed analyzing the fucosylation level of the carbohydrates without prior labeling (with 2-AB for example) and without the need to cleave the carbohydrates from the polypeptide chain (with PNGaseF) and correlated directly with the biological activity The analysis of fucosylation by Fcγ RIIIa chromatography is thus a direct, label- and modification-free method that avoids artefacts resulting from processing and modification steps and therefore results in higher and more reliable quality data The only preparation step is the removal of cell culture medium by Protein A purification We demonstrated, for the first time, that the Fcγ RIIIa affinity column can be used to enrich antibody species with different fucosylation levels Three antibody fractions were isolated by this procedure: antibodies enriched in fully fucosylated (1), monofucosylated (2) or a-fucosylated (3) carbohydrates Each antibody population was subjected to further analysis of its biological activity The mono- and a-fucosylated antibodies revealed a similar behavior in ADCC assay and SPR-assessed Fcγ RIIIa interaction These results demonstrated that one a-fucosylated glycan on the antibody is sufficient for increased affinity to Fcγ RIIIa, which is in line with the crystal structure of Fc with Fcγ RIIIa [12] showing that only one side of the Fc is binding to the Fcγ RIIIa As expected, the higher affinity of the a-fucosylated glycan dominates and de- termines the overall affinity of the antibody Consequently, 100% a-fucose content is not required to achieve enhanced effector functions, but 50% a-fucosylation content per IgG molecule is, theoretically, enough Among the different methods of glycoengineering that were developed, deletion of the fucosyltransferase [20] would allow reaching higher a-fucosylation levels than overexpressing recombinant wild-type β -1,4-N-acetyl-glucosaminyltransferase III and wild-type Golgi α -mannosidase II [19] However, according to the results shown here, this difference in a-fucosylation levels would not be reflected in ADCC activity and both methods can therefore be considered equally suitable to obtain highly active, glycoengineered antibodies With respect to the screening of clones for antibody production, our experiments imply that quantifying the ratio between double fucosylated and the mixture of mono- and a-fucosylated antibodies is sufficient As demonstrated here, the mono- and a-fucosylated antibodies both have similar biological activity and can therefore be equally selected To identify clones producing antibodies with low fucosylation levels, both steps of Protein A purification and Fcγ RIIIa chromatography were amenable to high-throughput analysis and were performed in this study in 96-well plates on an HPLC system The proportion of a-fucosylated antibodies in the analyzed samples, assessed by the presented chromatography method, correlated with the one obtained by MS analysis The Fcγ RIIIa chromatography analysis, combined with preceding protein A purification, enabled fast and efficient ranking of the antibody expressing clones for their titer and fucosylation level The duration of the chromatography run was optimized to min, which allowed automated screening in a 96-well format of large number of samples in a short time, representing a clear advantage of this approach over MS-analysis The Fcγ RIIIa construct was further optimized by dimerization through the introduction of an inert Fc-tag to increase production yields In addition, this construct contained an Avi-tag for specific, directed biotinylation and coupling of the receptor to Streptavidin sepharose This ensured that the receptor bioactivity was not affected in any way due to unspecific crosslinking In our experiments, only the mono- and a-fucosylated antibody species bound to the chemically coupled Fcγ RIIIa, which was likely due to impaired receptor accessibility In contrast, Fcγ RIIIa-Avi coupled via biotin efficiently interacted with both, fucosylated and afucosylated antibodies, which however differ in their affinity, as reflected in the elution pH The Fcγ RIIIa-Avi tagged, as well as the Fcγ RIIIa-his, allowed separating fully fucosylated antibodies A Freimoser–Grundschober, P Rueger and F Fingas et al / Journal of Chromatography A 1610 (2020) 460554 Table Amino acid sequence of the two Fcγ RIIIa constructs immobilized on the chromatographic matrices The biotinylated Fc fusion is the recommended construct for matrix preparation Human Fcγ RIIIa (V158) with C-terminal (Lysine)6- and (Histidine)6-fusion GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSW KNTALHKVTYLQNGKGRKYFHHNSDVYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGKKKKKKGHHHHHH Human Fcγ RIIIa (V158) with C-terminal AviTag, IgA Protease cleavage site and Fc fusion GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSW KNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGLNDIFEAQKIEWHELVVAPPAPEDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SL SPGK from the mono- and a-fucosylated species and to precisely determine the percentage of these two antibody groups Scoring these percentages allowed selecting the clones with strongest Fcγ RIIIa binding, which in turn correlated with higher biological activity in ADCC assays This analysis was performed successfully for 222 clones during the clone screening of a production cell line [21] Conclusion Preparative Fcγ RIIIa chromatography allowed to isolate three different fractions with antibodies enriched in fully fucosylated, mono-fucosylated or a-fucosylated carbohydrates The antibodies carrying mono- and a-fucosylated carbohydrates had equivalent increased biological activity, compared to the fully fucosylated ones Consequently, screening for at least one-fold a-fucosylated glycoengineered antibodies is sufficient during clone selection The recommended design for matrix preparation is the fusion of the extracellular domain of the Fcγ RIIIa V158 to an Fc with the P329G LALA mutation and an Avi-tag and immobilization of the biotinylated receptor to Streptavidin sepharose The resulting Fcγ RIIIa affinity chromatography enabled the separation of antibodies based on the fucosylation of their carbohydrates and/or the assessment of the fucosylation level of an antibody sample during clone screening It is a new, fast, stable, label- and modification-free efficient tool for both, preparative and analytical purposes, the read-out of which correlates directly with the biological activity of the analyzed antibodies Materials and methods 5.1 Expression of proteins 5.1.1 Expression and purification of soluble human Fcγ RIIIa(V158)K6H6 Human Fcγ RIIIa (V158) with C-terminal (Lysine)6- and (Histidine)6-fusion (Table 5) [22] was produced by calcium phosphate-transfection of HEK293-EBNA cells and harvested after days The secreted protein was purified via immobilized metal chelate chromatography (IMAC, NiNTA Superflow cartridge, Qiagen, Germany) using the manufacturer recommendations and polishing by size exclusion chromatography (HiLoad 16/60 Superdex 75; GE Healthcare, Sweden) with a mobile phase of mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3 , pH 7.4 5.1.2 Expression and purification of soluble human Fcγ RIIIa(V158)-Avi-IgA Protease-Fc fusion Human Fcγ RIIIa (V158) with C-terminal AviTag, IgA Protease cleavage site and Fc fusion (Table 5) was produced by transient transfection of HEK293 cells with 293-Free transfection reagent (Novagen) and harvested after days The secreted protein was purified via affinity chromatography using HiTrap MabSelectSuRe (GE Healthcare) and polishing by size exclusion chromatography on Su- perdex 200 (GE Healthcare) with a mobile phase of mM MOPS, 125 mM NaCl pH 7.2 5.1.3 Expression and purification of GEmAb and WTmAb GEmAb1, WTmAb1, GEmAb2 and GEmAb4 were produced in CHO cells in serum free medium (CD-CHO, Gibco) WTmAb4 and WTmAb5 were produced in HEK293F cells in serum free Opti-MEM I medium All IgGs were purified by affinity chromatography using HiTrap MabSelectSuRe (GE Healthcare) and size exclusion chromatography (HiLoad 16/60 Superdex 200; GE Healthcare, Sweden) WTmAb2 was produced in HEK EBNA cells in D-MEM with 10% FBS ultra-low IgG (Gibco) and purified under the same conditions with an additional wash at pH 5.5 to remove any bovine IgGs Supernatants from GEmAb3 were produced in CHO cells in serum free medium (CD-CHO, Gibco) 5.2 Matrix preparation and chromatography conditions 5.2.1 Preparation of Fcγ RIIIa(V158)-affinity matrix using NHS Sepharose FF for preparative purposes 30 mg Fcγ RIIIa(V158) were coupled to NHS activated Sepharose 4FF (GE Healthcare, Sweden) Shortly, Fcγ RIIIa(V158) was transferred into 0.2 M NaHCO3 , 0.5 M NaCl, pH 8.2 and incubated for h at room temperature with ml NHS activated beads, prewashed with mM cold HCl The reaction was quenched with 0.1 M Tris, pH 8.5 for h at room temperature The beads were then packed into a Tricorn 5/150 column (GE Healthcare) by gravity flow, followed by packing at 1.2 ml/min using an Äkta Explorer 10 (GE Healthcare), to a final column volume of 2.7 ml, at a column length of 14 cm 30 mg of human Fcγ RIIIa(V158) were immobilized in total Sepharose particle diameter is of 45-165 μm, pressure drop constraint was bar and loading capacity of final column was around mg antibody/ml column volume 5.2.2 Preparation of the affinity matrix using POROS AL for analytical purposes 10 mg Fcγ RIIIa(V158) in 0.1 M sodium phosphate, 0.05% (w/v) NaN3 , pH 7.0 were coupled to 0.14 g of dry POROS AL beads (Applied Biosystems, USA) with 0.1 M NaCNBH3 and overnight incubation at room temperature The reaction was quenched with M Tris, pH 7.4 and 50 mM NaCNBH3 for 30 at room temperature Finally, the beads were washed four times with 1M NaCl and three times with mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3 , pH 7.3 14 mg Fcγ RIIIa(V158) were coupled per gram of POROS AL beads ˚ POROS particle diameter is of 20 μm, pore diameter 50 0–10 0 A, pressure drop constraint max 170 bar, loading capacity of final column was 0.16 mg antibody/ml column volume 5.2.3 Preparation of the affinity matrix using streptavidin sepharose for analytical purposes An affinity column with Fcγ RIIIaV158 with avi-tag was prepared by in vitro biotinylation and subsequent coupling to Streptavidin sepharose This was done with the intact fusion polypeptide as well as with the receptor after having cleaved off the Fc-region 8 A Freimoser–Grundschober, P Rueger and F Fingas et al / Journal of Chromatography A 1610 (2020) 460554 5.2.3.1 Cleavage of Fc fusion protein by IgA protease (optional) The Fcγ RIIIa(V158) Avi-IgA-Fc construct was dyalized to 50 mM Tris pH and incubated with IgA Protease (Roche Diagnostics GmbH) at a ratio of w(protease)/w(fusion polypeptide) 1:100 at 21 °C overnight The Fc-tag was removed by HiTrap MabSelectSuRe (GE Healthcare) and the IgA protease by size exclusion chromatography on Superdex 75 (GE Healthcare) buffer and elution of samples was done with a linear pH gradient to 20 mM citrate, 150 mM NaCl pH 3.0 in 15 column volumes The experiments were carried out at room temperature The elution profile was obtained by continuous measurement of the absorbance at 280 nm 5.2.3.2 Biotinylation of receptor mg Fcγ RIIIaV158 or mg Fc tagged Fcγ RIIIaV158 in mM MOPS, 125 mM NaCl pH 7.2, 0.02% Tween20, and tablet Complete protease inhibitor (cOmplete ULTRA Tablets, Roche Diagnostics GmbH) in ml PBS were biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions (Bulk BIRA, Avidity LLC, Denver, CO, USA) Biotinylation reaction was done at room temperature overnight The modified protein was dialyzed against 20 mM sodium phosphate buffer comprising 150 mM NaCl, pH 7.5 at °C overnight to remove excess of biotin 5.3.1 ESI-MS analysis on Fc fragments or whole IgG Oligosaccharides were digested by EndoS and EndoH prior to ESI-MS analysis as described [23] 5.2.3.3 Coupling to streptavidin sepharose One gram Streptavidin Sepharose High Performance (GE Healthcare) was added to the biotinylated and dialyzed receptor and incubated for h while shaking and finally packed in a ml XK column (GE Healthcare) Streptavidin Sepharose particle diameter is of 34 μm with a loading capacity of biotinylated bovine serum albumin of mg/ml medium (according to the manufacturer) Loading capacity of the FcyRIIIa column was about mg antibody/ml column volume (with mg Fc dimerized FcyRIIIaV158 bound) 5.2.4 Conditions for preparative separation using Fcγ RIIIa(V158) immobilized on NHS Sepharose FF The chromatography column was equilibrated with 10 column volumes of 20 mM Tris, 20 mM MOPS, 20 mM sodium citrate, 100 mM NaCl, pH 8.0, followed by load of mg of purified antibody at a flow rate of 0.1 ml/min The column was washed with CV of 20 mM Tris, 20 mM MOPS, 20 mM sodium citrate, 100 mM NaCl, pH 8.0 The elution was carried out with a three step gradient of pH steps 4.6, 4.2 and 3.0 The peaks were collected, concentrated and purified by protein A 5.2.5 Conditions for analytical chromatography using Fcγ RIIIa(V158) immobilized on POROS AL POROS AL beads with Fcγ RIIIa(V158) were packed in a × 20 mm Upchurch Scientific column (column volume: 60 μl) which was mounted in the Agilent 1200 HPLC system (Agilent Technologies, USA) 10 mM Tris, 50 mM glycine, 100 mM NaCl, pH 8.0 was used to equilibrate and wash the column; the elution was carried out with 10 mM Tris, 50 mM glycine, 100 mM NaCl, pH 3.0 (flow rate 0.5 ml/min) At time zero the antibody preparation (10 μg) was injected and washed for min, then eluted in a step gradient of 0.66 min, before re-equilibration for 4.33 For high throughput analysis of IgGs from supernatants the samples were first purified using Protein A on the Agilent 1200 HPLC system and collected in a 96-well plate The samples were neutralized by adding 1:40 v/v M Tris pH 8.0 and either reinjected on the Fcγ RIIIa(V158) chromatography column (injection volumes adjusted to inject 10 μg) or were digested with PNGase F for MALDI TOF MS analysis of the carbohydrates 5.2.6 Conditions for analytical chromatography using Fcγ RIIIa(V158)-Fc immobilized on Streptavidin sepharose Antibody samples containing 30 to 50 μg of protein were diluted at a volume ratio of 1:1 with equilibration buffer, 20 mM citric acid/150 mM NaCl pH 6.0, and applied to the Fcγ RIIIa column The column was washed with 2.5 column volumes of equilibration 5.3 Analytical methods for analyses of carbohydrate composition 5.3.2 MALDI-TOF analysis on released carbohydrates The N-linked oligosaccharides were cleaved of the purified IgGs by incubation with 0.005 U of PNGase F (QAbio, USA) and EndoH (QAbio, USA) in 20 mM Tris pH 8.0 at 37 °C for 16 h This resulted in free oligosaccharides that were analyzed by mass spectrometry (Autoflex, Bruker Daltonics GmbH) in positive ion mode according to [24] 5.3.3 2-AB (aminobenzamide) labeling and RP-UPLC of carbohydrates for the determination of the relative oligosaccharide distribution The content of a-fucosylated N-glycans was determined by glycan release with PNGaseF, followed by derivatization with 2aminobenzamidine and separation by RP-UPLC Glycans were released by PNGaseF (New England Biolabs) digestion overnight at 37° After addition of final 20 mM acetic acid and incubation for 15 at 45 °C, 2-AB labeling of the released glycans was performed with the GlycoProfile 2-AB labeling kit (Sigma) according to the manufacturer’s instructions Fucosylated and non-fucosylated labeled glycans were separated by a linear gradient of 0.1% aqueous formic acid to 0.1% formic acid in acetonitrile on an Acquity HSS T3 column (Thermo Scientific) at 80° and detected by fluorescence 5.4 Surface plasmon resonance SPR interaction analysis was performed on a Biacore T100 system (GE Healthcare) Human antigen for mAb2 was immobilized by amine coupling on a CM5 chip using the manufacturer’s instructions (GE Healthcare) The IgG fractions were captured for 90 s at 100 nM and 10 μl/min The human Fcγ RIIIa(V158) was injected as analyte at a concentration range from 1.95–500 nM and a flow rate of 50 μl/min for 120 s The dissociation is monitored for 220 s The surface was regenerated by two injections of 10 mM glycine, pH 2.0 for 60 s Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell Association rates (kon ) and dissociation rates (koff ) were calculated using a one-to-one Langmuir binding model with RI = and Rmax = local (BIACORE R T100 Evaluation Software version 1.1.1) by simultaneously fitting the association and dissociation sensorgrams The equilibrium dissociation constant (KD) was calculated as the ratio koff /kon 5.5 ADCC Raji (for mAb1 ADCC assay) or A549 (for mAb2 ADCC assay) cells were harvested (adherent cells with Trypsin/EDTA), washed and labeled for 30 at 37 °C with Calcein (Invitrogen) After 30 min, cells were washed times with AIM V and re-suspended in AIM V medium Subsequently, they were plated in a roundbottom, 96-well plate at a concentration of 30,0 0 cells/well The respective antibody dilutions were added and incubated for 10 before contact with human effector cells (NK92 1708 clone LC3 E11 = NK92 cells transfected with Fcγ RIIIa(V158)) Effector and target cells were co-incubated at a ratio of 3:1 for h at 37 °C A Freimoser–Grundschober, P Rueger and F Fingas et al / Journal of Chromatography A 1610 (2020) 460554 Lactate dehydrogenase (LDH) release was measured using the LDH Cytotoxicity detection Kit (Roche Applied Science) The calcein retention was measured by lysing the remaining cells with borate buffer (5 mM borate + 0.1% v/v Triton X100) followed by measurement of the calcein fluorescence For calculation of antibodydependent killing, spontaneous release (only target + effector cells without antibody) was set to 0% killing and maximal release (target cells + 2% v/v Triton X-100) was set to 100% killing [10] [11] [12] Declaration of Competing Interest [13] None Acknowledgments [14] We thank Hans Koll for the analysis of the repartition of carbohydrates on whole Fc fragments Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors All authors are (or were) Roche employees (at the time when the study was conducted) 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Papac, A Wong, A.J Jones, Analysis of acidic oligosaccharides and glycopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Anal Chem 68 (1996) 3215–3223  ... system The proportion of a-fucosylated antibodies in the analyzed samples, assessed by the presented chromatography method, correlated with the one obtained by MS analysis The Fcγ RIIIa chromatography. .. with the P329G LALA mutation and an Avi-tag and immobilization of the biotinylated receptor to Streptavidin sepharose The resulting Fcγ RIIIa affinity chromatography enabled the separation of antibodies. .. cleavage site and the Fc region of an IgG1, which included the P329G, L234A and L235A amino acid substitutions to avoid interactions of the Fc part with the Fcγ RIIIa [18] The Fcγ RIIIa- Avi-Fc