Correlated analytical and functional evaluation of higher order structure perturbations from oxidation of NISTmAb.

The clinical efficacy and safety of protein-based drugs such as monoclonal antibodies (mAbs) rely on the integrity of the protein higher order structure (HOS) during product development, manufacturing, storage, and patient administration. As mAb-based drugs are becoming more prevalent in the treatment of many illnesses, the need to establish metrics for quality attributes of mAb therapeutics through high-resolution techniques is also becoming evident. To this end, here we used a forced degradation method, time-dependent oxidation by hydrogen peroxide, on the model biotherapeutic NISTmAb and evaluated the effects on HOS with orthogonal analytical methods and a functional assay. To monitor the oxidation process, the experimental workflow involved incubation of NISTmAb with hydrogen peroxide in a benchtop nuclear magnetic resonance spectrometer (NMR) that followed the reaction kinetics, in real-time through the water proton transverse relaxation rate R2(1H2O). Aliquots taken at defined time points were further analyzed by high-field 2D 1H-13C methyl correlation fingerprint spectra in parallel with other analytical techniques, including thermal unfolding, size-exclusion chromatography, and surface plasmon resonance, to assess changes in stability, heterogeneity, and binding affinities. The complementary measurement outputs from the different techniques demonstrate the utility of combining NMR with other analytical tools to monitor oxidation kinetics and extract the resulting structural changes in mAbs that are functionally relevant, allowing rigorous assessment of HOS attributes relevant to the efficacy and safety of mAb-based drug products.

Ligand-Bound Forced Degradation as a Strategy to Generate Functionally Relevant Analytical Challenge Materials for Assessment of CQAs.

Therapeutic monoclonal antibodies (mAbs) contain a variety of amino acids that are susceptible to enzymatic, chemical, and physical modifications. These modifications can happen throughout production, purification, formulation, and storage and many are known to affect the biological activity of a mAb. Methods that are able to characterize and evaluate these attributes are critical in order to understand how they might alter biological activity. Methods capable of site-specific monitoring of these critical quality attributes are extremely valuable to biopharmaceutical research but also require well-defined materials with site-specific attribute modifications. Here, we describe the development and application of a strategy to generate functionally relevant analytical challenge materials that have unique site-specific attributes. This method involves the use of a ligand that is bound to the mAb during oxidative stress resulting in unique oxidation patterns with some methionine residues protected while others are exposed to oxidation. These unique materials were used to develop a rapid surface plasmon resonance (SPR) assay that could detect methionine oxidation in both the Fab and Fc regions using specific molecular probes. The addition of uniquely oxidized materials to our data set enabled us to determine specific methionine residues vital to binding. Further analysis showed that antibody oxidation could also be rapidly detected in multiple domains from qualitative thermal melting using intrinsic tryptophan fluorescence. Methionine oxidation of an antibody was explored in this study, but we envision this method could be useful to explore structure function relationships of a variety of antibody modifications and modifications to other biologically relevant protein drugs.

Principal component analysis for automated classification of 2D spectra and interferograms of protein therapeutics: influence of noise, reconstruction details, and data preparation.

Protein therapeutics have numerous critical quality attributes (CQA) that must be evaluated to ensure safety and efficacy, including the requirement to adopt and retain the correct three-dimensional fold without forming unintended aggregates. Therefore, the ability to monitor protein higher order structure (HOS) can be valuable throughout the lifecycle of a protein therapeutic, from development to manufacture. 2D NMR has been introduced as a robust and precise tool to assess the HOS of a protein biotherapeutic. A common use case is to decide whether two groups of spectra are substantially different, as an indicator of difference in HOS. We demonstrate a quantitative use of principal component analysis (PCA) scores to perform this decision-making, and demonstrate the effect of acquisition and processing details on class separation using samples of NISTmAb monoclonal antibody Reference Material subjected to two different oxidative stress protocols. The work introduces an approach to computing similarity from PCA scores based upon the technique of histogram intersection, a method originally developed for retrieval of images from large databases. Results show that class separation can be robust with respect to random noise, reconstruction method, and analysis region selection. By contrast, details such as baseline distortion can have a pronounced effect, and so must be controlled carefully. Since the classification approach can be performed without the need to identify peaks, results suggest that it is possible to use even more efficient measurement strategies that do not produce spectra that can be analyzed visually, but nevertheless allow useful decision-making that is objective and automated.

Comparative Analysis of One-Dimensional Protein Fingerprint by Line Shape Enhancement and Two-Dimensional 1H,13C Methyl NMR Methods for Characterization of the Higher Order Structure of IgG1 Monoclonal Antibodies.

The use of NMR spectroscopy has emerged as a premier tool to characterize the higher order structure of protein therapeutics and in particular IgG1 monoclonal antibodies (mAbs). Due to their large size, traditional 1H-15N correlation experiments have proven exceedingly difficult to implement on mAbs, and a number of alternative techniques have been proposed, including the one-dimensional (1D) 1H protein fingerprint by line shape enhancement (PROFILE) method and the two-dimensional (2D) 1H-13C methyl correlation-based approach. Both 1D and 2D approaches have relative strengths and weaknesses, related to the inherent sensitivity and resolution of the respective methods. To further increase the utility of NMR to the biopharmaceutical community, harmonized criteria for decision making in employing 1D and 2D approaches for mAb characterization are warranted. To this end, we have conducted an interlaboratory comparative study of the 1D PROFILE and 2D methyl methods on several mAbs samples to determine the degree to which each method is suited to detect spectral difference between the samples and the degree to which results from each correlate with one another. Results from the study demonstrate both methods provide statistical data highly comparable to one another and that each method is capable of complementing the limitations commonly associated with the other, thus providing a better overall picture of higher order structure.

Glycoengineering of Antibodies for Modulating Functions.

Antibodies are immunoglobulins that play essential roles in immune systems. All antibodies are glycoproteins that carry at least one or more conserved N-linked oligosaccharides (N-glycans) at the Fc domain. Many studies have demonstrated that both the presence and fine structures of the attached glycans can exert a profound impact on the biological functions and therapeutic efficacy of antibodies. However, antibodies usually exist as mixtures of heterogeneous glycoforms that are difficult to separate in pure glycoforms. Recent progress in glycoengineering has provided useful methods that enable production of glycan-defined and site-selectively modified antibodies for functional studies and for improved therapeutic efficacy. This review highlights major approaches in glycoengineering of antibodies with a focus on recent advances in three areas: glycoengineering through glycan biosynthetic pathway manipulation, glycoengineering through in vitro chemoenzymatic glycan remodeling, and glycoengineering of antibodies for site-specific antibody-drug conjugation.

Synthetic HIV V3 Glycopeptide Immunogen Carrying a N334 N-Glycan Induces Glycan-Dependent Antibodies with Promiscuous Site Recognition.

The N332 high-mannose glycan on the HIV-1 gp120 V3-loop is the target of many bNAbs. About 17% HIV isolates carry the N332 to N334 mutation, but the antibody recognition of the N334 N-glycan and its immunogenicity are not well characterized. Here we report the chemoenzymatic synthesis, antigenicity, and immunogenicity of the V3 N334 glycopeptides from HIV-1 A244 gp120, a key component in the partially successful Thai clinical trials. We found that synthetic V3 glycopeptide carrying a N334 high-mannose glycan could be recognized by bNAb PGT128 and PGT126 but not by 10-1074. Rabbit immunization with the synthetic three-component A244 glycopeptide immunogen elicited substantial glycan-dependent antibodies with broad reactivity to various HIV-1 gp120/gp140 carrying N332 or N334 glycosylation sites. These results indicated that the N334 site is vulnerable and the A244 V3 glycopeptide represents a valuable immunogen for further HIV-1 vaccine studies.

Site-selective chemoenzymatic glycoengineering of Fab and Fc glycans of a therapeutic antibody.

The N-glycans attached to the Fab and Fc domains play distinct roles in modulating the functions of antibodies. However, posttranslational site-selective modifications of glycans in antibodies and other multiply glycosylated proteins remain a challenging task. Here, we report a chemoenzymatic method that permits independent manipulation of the Fab and Fc N-glycans, using cetuximab as a model therapeutic monoclonal antibody. Taking advantage of the substrate specificity of three endoglycosidases (Endo-S, Endo-S2, and Endo-F3) and their glycosynthase mutants, together with an unexpected substrate site-selectivity of a bacterial α1,6-fucosidase from Lactobacillus casei (AlfC), we were able to synthesize an optimal homogeneous glycoform of cetuximab in which the heterogeneous and immunogenic Fab N-glycans were replaced with a single sialylated N-glycan, and the core-fucosylated Fc N-glycans were remodeled with a nonfucosylated and fully galactosylated N-glycan. The glycoengineered cetuximab demonstrated increased affinity for the FcγIIIa receptor and significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

Heterologous recombinant expression of non-originator NISTmAb.

The successful development and regulatory approval of originator and biosimilar therapeutic proteins requires a systems approach to upstream and downstream processing as well as product characterization and quality control. Innovation in process design and control, product characterization strategies, and data integration represent an ecosystem whose concerted advancement may reduce time-to-market and further improve comparability and biosimilarity programs. The biopharmaceutical community has made great strides to this end, yet there currently exists no pre-competitive monoclonal antibody (mAb) expression platform for open innovation. Here, we describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. It was found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy, intact mass spectrometry, and surface plasmon resonance were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.

Multivalent Antigen Presentation Enhances the Immunogenicity of a Synthetic Three-Component HIV-1 V3 Glycopeptide Vaccine.

HIV-1 envelope glycoproteins gp120 and gp41 are presented on the virus surface as a trimer of heterodimer and are the targets of broadly neutralizing antibodies (bNAbs). We describe here the synthesis and preliminary immunological evaluation of a three-component trivalent HIV-1 V3 glycopeptide immunogen aiming to raise glycopeptide epitope-specific antibodies. Click chemistry confers efficient synthesis of the lipopeptide-glycopeptide conjugate that carries three copies of HIV-1 JR-FL gp120 V3 glycopeptide with a high-mannose glycan at the N332 glycosylation site. We found that the multivalent presentation substantially enhanced the immunogenicity of the V3 glycopeptide. The antisera induced by the three-component multivalent glycopeptide immunogen exhibited stronger binding to heterologous HIV-1 gp120s and the trimeric gp140s than that from the monovalent glycopeptide immunogen. The antisera generated from this preliminary rabbit immunization did not show virus neutralization activity, probably due to the lack of somatic maturation. The ability to elicit substantial glycopeptide epitope-specific antibodies by the three-component trivalent glycopeptide immunogen suggests that it could serve as a valuable vaccine component in combination with other vaccine candidates for further immunization studies.

Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies.

N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or ß-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics' glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies.

Synthetic Three-Component HIV-1 V3 Glycopeptide Immunogens Induce Glycan-Dependent Antibody Responses.

Eliciting broadly neutralizing antibody (bNAb) responses against HIV-1 is a major goal for a prophylactic HIV-1 vaccine. One approach is to design immunogens based on known broadly neutralizing epitopes. Here we report the design and synthesis of an HIV-1 glycopeptide immunogen derived from the V3 domain. We performed glycopeptide epitope mapping to determine the minimal glycopeptide sequence as the epitope of V3-glycan-specific bNAbs PGT128 and 10-1074. We further constructed a self-adjuvant three-component immunogen that consists of a 33-mer V3 glycopeptide epitope, a universal T helper epitope P30, and a lipopeptide (Pam3CSK4) that serves as a ligand of Toll-like receptor 2. Rabbit immunization revealed that the synthetic self-adjuvant glycopeptide could elicit substantial glycan-dependent antibodies that exhibited broader recognition of HIV-1 gp120s than the non-glycosylated V3 peptide. These results suggest that the self-adjuvant synthetic glycopeptides can serve as an important component to elicit glycan-specific antibodies in HIV vaccine design.

Synthetic multivalent V3 glycopeptides display enhanced recognition by glycan-dependent HIV-1 broadly neutralizing antibodies.

We describe here the synthesis of novel multivalent HIV V3 domain glycopeptides and their binding to broadly neutralizing antibodies PGT128 and 10-1074. Our binding data reveal a distinct mode of antigen recognition by the two antibodies and further suggest that multivalent glycopeptides could mimic the neutralizing epitopes more efficiently than the monomeric glycopeptide.

Systematic Synthesis and Binding Study of HIV V3 Glycopeptides Reveal the Fine Epitopes of Several Broadly Neutralizing Antibodies.

A class of new glycan-reactive broadly neutralizing antibodies represented by PGT121, 10-1074, and PGT128 has recently been discovered that targets specific N-glycans and the peptide region around the V3 domain. However, the glycan specificity and fine epitopes of these bNAbs remain to be further defined. We report here a systematic chemoenzymatic synthesis of homogeneous V3 glycopeptides derived from the HIV-1 JR-FL strain carrying defined N-glycans at N332, N301, and N295 sites. Antibody binding studies revealed that both the nature and site of glycosylation in the context of the V3 domain were critical for high-affinity binding. It was found that antibody PGT128 exhibited specificity for high-mannose N-glycan with glycosylation site promiscuity, PGT121 showed binding specificity for glycopeptide carrying a sialylated N-glycan at N301 site, and 10-1074 was specific for glycopeptide carrying a high-mannose N-glycan at N332 site. The synthesis and binding studies permit a detailed assessment of the glycan specificity and the requirement of peptide in the context of antibody-antigen recognition. The identified glycopeptides can be used as potential templates for HIV vaccine design.

Modulating IgG effector function by Fc glycan engineering.

IgG antibodies contain a conserved N-glycosylation site on the Fc domain to which a complex, biantennary glycan is attached. The fine structures of this glycan modulate antibody effector functions by affecting the binding affinity of the Fc to diverse Fc receptor family members. For example, core fucosylation significantly decreases antibody-dependent cellular cytotoxicity (ADCC), whereas terminal α2,6-sialylation plays a critical role in the anti-inflammatory activity of human i.v. immunoglobulin therapy. The effect of specific combinations of sugars in the glycan on ADCC remains to be further addressed, however. Therefore, we synthesized structurally well-defined homogeneous glycoforms of antibodies with different combinations of fucosylation and sialylation and performed side-by-side in vitro FcγR-binding analyses, cell-based ADCC assays, and in vivo IgG-mediated cellular depletion studies. We found that core fucosylation exerted a significant adverse effect on FcγRIIIA binding, in vitro ADCC, and in vivo IgG-mediated cellular depletion, regardless of sialylation status. In contrast, the effect of sialylation on ADCC was dependent on the status of core fucosylation. Sialylation in the context of core fucosylation significantly decreased ADCC in a cell-based assay and suppressed antibody-mediated cell killing in vivo. In contrast, in the absence of fucosylation, sialylation did not adversely impact ADCC.

An Fc-Small Molecule Conjugate for Targeted Inhibition of the Adenosine†2A Receptor.

The adenosine A2A receptor (A2A R) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A2A R have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well-established potent and selective small molecule A2A R antagonist, ZM-241385 (ZM), has a short pharmacokinetic half-life and the potential for systemic toxicity due to A2A R effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein-small molecule conjugate, Fc-ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc-ZM was a potent A2A R antagonist, as measured by a cell-based cAMP assay. Cell-based assays also revealed that Fc-ZM could stimulate interferon γ production in splenocytes in a fashion that was dependent on the presence of A2A R. We found that Fc-ZM, compared with the small molecule ZM, was a superior A2A R antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule.

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling.

Glycosylation can exert a profound impact on the structures and biological functions of antibodies. Glycosylation remodeling using the endoglycosidase-catalyzed deglycosylation and transglycosylation approach is emerging as a promising platform to produce homogeneous glycoforms of antibodies, but the broad application of this method will require the availability of highly efficient glycosynthase mutants. We describe in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) and the evaluation of the resulting mutants for their hydrolysis and transglycosylation activities. We found that mutations at the Asp-184 residue gave mutants that demonstrated significantly different properties, some possessed potent transglycosylation activity with diminished hydrolysis activity but others did not, which would be otherwise difficult to predict without the comparative study. In contrast to the previously reported Endo-S mutants that are limited to action on complex type N-glycans, the Endo-S2 glycosynthases described here, including D184M and D184Q, were found to have remarkably relaxed substrate specificity and were capable of transferring three major types (complex, high-mannose, and hybrid type) of N-glycans for antibody glycosylation remodeling. In addition, the Endo-S2 glycosynthase mutants were found to be much more active in general than the Endo-S mutants for transglycosylation. The usefulness of these Endo-S2 glycosynthase mutants was exemplified by an efficient glycosylation remodeling of two therapeutic monoclonal antibodies, rituximab and trastuzumab (Herceptin).

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins.

Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptorsubstrate specificity toward α1,6-fucosyl-GlcNAc-Asn or α1,6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi- and triantennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize triantennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly, the new triantennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first endoglycosidase-based glycosynthases capable of transferring triantennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.

Evaluation of a glycoengineered monoclonal antibody via LC-MS analysis in combination with multiple enzymatic digestion.

Glycosylation affects the efficacy, safety and pharmacokinetics/pharmacodynamics properties of therapeutic monoclonal antibodies (mAbs), and glycoengineering is now being used to produce mAbs with improved efficacy. In this work, a glycoengineered version of rituximab was produced by chemoenzymatic modification to generate human-like N-glycosylation with α 2,6 linked sialic acid. This modified rituximab was comprehensively characterized by liquid chromatography-mass spectrometry and compared to commercially available rituximab. As anticipated, the majority of N-glycans were converted to α 2,6 linked sialic acid, in contrast to CHO-produced rituximab, which only contains α 2,3 linked sialic acid. Typical posttranslational modifications, such as pyro-glutamic acid formation at the N-terminus, oxidation at methionine, deamidation at asparagine, and disulfide linkages were also characterized in both the commercial and glycoengineered mAbs using multiple enzymatic digestion and mass spectrometric analysis. The comparative study reveals that the glycoengineering approach does not cause any additional posttranslational modifications in the antibody except the specific transformation of the glycoforms, demonstrating the mildness and efficiency of the chemoenzymatic approach for glycoengineering of therapeutic antibodies.

Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity.

IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

Chemoenzymatic Glyco-engineering of Monoclonal Antibodies.

Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications.