RESUMEN
Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.
RESUMEN
Adeno-associated viruses (AAVs) are useful vehicles for gene therapy because of their stability, low immunogenicity. and non-pathogenicity. However, disparity in AAV sample preparations (e.g., in capsid composition, DNA packaging, and impurities) gives rise to product heterogeneity, with possibly undesired effects on gene delivery. Ideally, AAV production should be with full control of AAV structure and genetic payload. Therefore, robust, efficient, and low material consuming methods are essential to characterize AAVs. Here, we use two emerging single-molecule techniques, mass photometry and Orbitrap-based charge-detection mass spectrometry, and show how they may efficiently and accurately characterize AAVs. We were able to resolve heterogeneous pools of particles, evaluating AAVs from two different serotypes (AAV8 and AAV2), produced by three independent production platforms, either lacking a genome or packed with a transgene. Together our data confirm that the different AAV production methods result in rather different and diverse AAV particle distributions. Especially for the packed AAVs, frequently additional subspecies were observed, next to the expected packed genome, mostly resulting from under- or overpackaging of genome material and/or residual empty particles. This work further establishes that both these single-particle techniques may become valuable tools in characterizing AAVs before they are used in gene therapy.
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A relatively low clearance is one of the prominent favorable features of immunoglobulin G1-based therapeutic monoclonal antibodies (mAbs). Various studies have observed differential clearance of mAb glycoforms, including oligomannose glycoforms, which are considered a critical quality attribute because they show higher clearance than complex type glycoforms. Glycoform clearance, however, has not previously been studied after subcutaneous injection or in a porcine model system. Here, we performed glycoform-resolved pharmacokinetic (PK) analysis of two mAbs in Göttingen minipigs. We found glycoform effects on clearance to be largely the same for subcutaneous and intravenous injection and in line with observations in other species. Oligomannose glycoforms were cleared up to 25% faster and monoantennary glycoforms up to 8% faster than agalactosylated complex glycoforms. Sialylated glycoforms were cleared at approximately the same rate as fully galactosylated glycoforms. Importantly, we report here an impact of galactosylation on the PK of a mAb for the first time. Whether increased galactosylation led to slower or faster clearance seemed to depend on the overall glycosylation profile. When clearance of galactosylated glycoforms was slower, the mAb showed higher galactosylation in serum at maximum concentration after subcutaneous injection compared to both intravenous injection and the injected material. Whether this higher galactosylation after subcutaneous injection has consequences for therapeutic efficacy remains to be investigated. In conclusion, preferential clearance of antibody glycoforms can be simulated in the minipig model with intravenous as well as subcutaneous injections. Furthermore, we observed a glycoform bias in the absorption from skin into circulation after subcutaneous injection based on galactosylation.Abbreviations: AUC - area under the curve; CL/F - apparent clearance as a function of bioavailability following SC administration; Cmax - maximum serum concentration; CQA critical quality attribute; FcγR - Fc gamma receptor; IgG - immunoglobulin G; IV - intravenous; LC-MS - liquid chromatography - mass spectrometry; mAb - therapeutic monoclonal antibody; PK - pharmacokinetics; SC - subcutaneous; TMDD - target-mediated drug disposition.
Asunto(s)
Anticuerpos Monoclonales , Inmunoglobulina G , Animales , Porcinos , Inyecciones Intravenosas , Porcinos Enanos/metabolismo , Inmunoglobulina G/metabolismo , Glicosilación , Inyecciones SubcutáneasRESUMEN
In vitro glycoengineering using exoenzymes for specific modification is recognized as appropriate method to tailor sugar moieties of glycan structures during the recombinant production of monoclonal antibodies (mAbs). This report describes enhanced in vitro glycoengineering approaches using ß1,4-galactosyltransferase and α2,6-sialyltransferase to improve the efficiency of galactosylation and sialylation with the aim to implement in vitro glycoengineering into common mAb purification processes. Feasibility studies tested the potential of different in vitro glycoengineering protocols (two-step vs. one-step) to facilitate the overall procedure. Scalability of the reactions was demonstrated for mAb amounts ranging from 1 mg to 1 g. Additionally, the reactions of ß1,4-galactosyltransferase and α2,6-sialyltransferase were shown to work on column during affinity chromatography using Protein A or KappaSelect, the latter providing more efficient galactosylation and sialylation of IgG1 and IgG4 mAbs. Performing in vitro glycoengineering on column enabled the use of cell culture harvest that yielded results comparable to those of purified bulk. Based thereon, an optimized two-step mixed mode approach was found most appropriate to integrate in vitro glycoengineering of the IgG1 mAb into the overall manufacturing process. Using harvest for on-column reaction of ß1,4-galactosyltransferase combined with in-solution reaction of α2,6-sialyltransferase, this approach yielded 100% biantennary galactosylation and 61% biantennary sialylation. Moreover, the enzymes applied in in vitro glycoengineering could be separated, recycled and reused in further reactions to improve economic efficiency. Overall, the study provides a toolbox for in vitro glycoengineering and presents an optimized easy-to-handle workflow to implement this method into the downstream processing of industrial mAb production.
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Anticuerpos Monoclonales , Sialiltransferasas , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Galactosiltransferasas/metabolismo , Glicosilación , Inmunoglobulina G/química , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
Good pharmacokinetic (PK) behavior is a key prerequisite for sufficient efficacy of therapeutic monoclonal antibodies (mAbs). Fc glycosylation is a critical quality attribute (CQA) of mAbs, due to its impact on stability and effector functions. However, the effects of various IgG Fc glycoforms on antibody PK remain unclear. We used a combination of glycoengineering and glycoform-resolved PK measurements by mass spectrometry (MS) to assess glycoform effects on PK. Four differently glycoengineered mAbs, each still containing multiple glycoforms, were separately injected into rats. Rat models have been shown to be predictive of human PK. At different time points, blood was taken, from which the mAbs were purified and analyzed with a liquid chromatography-MS-based bottom-up glycoproteomics approach. This allowed us to follow changes in the glycosylation profiles of each glycoengineered mAb over time. Enzyme-linked immunosorbent assay measurements provided an absolute concentration in the form of a sum value for all glycoforms. Information from both readouts was then combined to calculate PK parameters per glycoform. Thereby, multiple glycoform kinetics were resolved within one mAb preparation. We confirmed increased clearance of high-mannose (Man5) and hybrid-type (Man5G0) glycoforms. Specifically, Man5 showed a 1.8 to 2.6-fold higher clearance than agalactosylated, complex glycans (G0F). Unexpectedly, clearance was even higher (4.7-fold) for the hybrid-type glycan Man5G0. In contrast, clearance of agalactosylated, monoantennary glycoforms (G0F-N) was only slightly increased over G0F (1.2 to 1.4-fold). Thus, monoantennary, hybrid-type and high-mannose glycoforms should be distinguished in CQA assessments. Strikingly, α2,3-linked sialylation did not affect clearance, contradicting the involvement of the asialoglycoprotein receptor in mAb clearance.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Cromatografía Liquida/métodos , Glicopéptidos/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Espectrometría de Masas/métodos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Ensayo de Inmunoadsorción Enzimática , Glicopéptidos/inmunología , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Manosa/metabolismo , Polisacáridos/metabolismo , Ingeniería de Proteínas/métodos , Proteómica/métodos , Ratas WistarRESUMEN
Minor changes in the quality of biologically manufactured monoclonal antibodies (mAbs) can affect their bioactivity and efficacy. One of the most important variations concerns the N-glycosylation pattern, which directly affects an anti-tumor mechanism called antibody-dependent cell-meditated cytotoxicity (ADCC). Thus, careful engineering of mAbs is expected to enhance both protein-receptor binding and ADCC. The specific aim of this study is to evaluate the influence of terminal carbohydrates within the Fc region on the interaction with the FcγRIIIa/CD16a receptor in native and label-free conditions. The single mAb molecule comprises variants with minimal and maximal galactosylation, as well as α2,3 and α2,6-sialic acid isomers. Here, we apply native electrospray ionization mass spectrometry to determine the solution-phase antibody-receptor equilibria and by using temperature-controlled nanoelectrospray, a thermal stability of the complex is examined. Based on these, we prove that the galactosylation of a fucosylated Fc region increases the binding to CD16a 1.5-fold when compared with the non-galactosylated variant. The α2,6-sialylation has no significant effect on the binding, whereas the α2,3-sialylation decreases it 1.72-fold. In line with expectation, the galactoslylated and α2,6-sialylated mAb:CD16a complex exhibit higher thermal stability when measured in the temperature gradient from 20 to 50°C. The similar binding pattern is observed based on surface plasmon resonance analysis and immunofluorescence staining using natural killer cells. The results of our study provide new insight into N-glycosylation-based interaction of the mAb:CD16a complex.
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Anticuerpos Monoclonales/química , Ingeniería Celular/métodos , Fragmentos Fc de Inmunoglobulinas/química , Receptores de IgG/inmunología , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Glicosilación , HumanosRESUMEN
The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure-function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.
RESUMEN
Aim: Fragment crystallizable (Fc) glycosylation of immunoglobulin G-type monoclonal antibodies applied to therapeutic applications is regarded a critical quality attribute and can influence bioactivity, pharmacokinetics and/or immunogenicity/safety. Investigating the impact of certain Fc N-glycans is therefore of importance to assess its criticality for a therapeutic product. This has been done for N-glycan types like fucosylation, galactosylation or sialylation. There were contradictory results reported for functionality especially with regard to sialylation. Material & methods: We elucidated the effect of terminal sialic acid residues on Fcγ receptor binding and antibody dependent cytotoxicity activity of two immunoglobulin G1 antibodies with different levels of fucosylation/bi-secting. Conclusion: We found the impact to be specific to the sialylation linkage type, in other words, α2,3- versus α2,6-linked sialic acid attached to the terminal galactose residues.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Fucosa/metabolismo , Inmunoglobulina G/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores de IgG/metabolismo , Glicosilación , Humanos , Ácidos Neuramínicos/química , Ácidos Neuramínicos/metabolismo , Unión ProteicaRESUMEN
Oxidation of monoclonal therapeutic antibodies (mAbs) can affect binding to Fc-receptors and potentially influence pharmacokinetics or effector functions like e.g. antibody dependent cellular phagocytosis (ADCP). Recently, it has been demonstrated that binding to FcγRIIa (H131) is affected by methionine oxidation of the Fc-portion but it is currently unknown which methionine is responsible for decreased binding. We separated an oxidized IgG1 monoclonal antibody based on the oxidation state of methionine 252 and analyzed fractionated material in receptor binding experiments as well as in functional (cell-based) assays. Although the unfractionated mixture demonstrated weaker interaction/activation of the receptor, differently oxidized isolated subspecies can lead both to stronger as well as weaker binding and activation of the histidine variant of FcγRIIa.
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Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/inmunología , Metionina/inmunología , Receptores de IgG/inmunología , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos/inmunología , Cromatografía Liquida/métodos , Histidina/genética , Histidina/inmunología , Histidina/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Espectrometría de Masas/métodos , Metionina/metabolismo , Oxidación-Reducción , Unión Proteica , Receptores de IgG/genética , Receptores de IgG/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
The therapeutic activity of monoclonal antibodies can involve immune cell mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC), an activity that is modulated by the structure of Fc-glycans, and in particular the lack of core fucose. The heterogeneity of these glycostructures and the inherent variability of traditional PBMC-based in vitro ADCC assays, have made it challenging to quantitatively assess the impact of other glycostructures on ADCC activity. We applied a quantitative NK cell based assay to generate a database consisting of Fc-glycostructure and ADCC data from 54 manufacturing batches of a CHO-derived monoclonal antibody. Explorative analysis of the data indicated that, apart from afucosylation, galactosylation levels could influence ADCC activity. We confirmed this hypothesis by demonstrating enhanced ADCC upon enzymatic hypergalactosylation of four different monoclonal antibodies derived using standard CHO manufacturing processes. Furthermore we quantitatively compare the effects of galactosylation and afucosylation in the context of glycan heterogeneity and demonstrate that while galactose can influence ADCC activity, afucosylation remains the primary driver of this activity.
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Anticuerpos Monoclonales/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Galactosa/metabolismo , Inmunoglobulina G/metabolismo , Receptores de IgG/inmunología , Anticuerpos Monoclonales/química , Fucosa/metabolismo , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Células Asesinas Naturales/inmunología , Receptores de IgG/química , Receptores de IgG/metabolismoRESUMEN
Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human ß-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Km for N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.
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Sialiltransferasas/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Clonación Molecular , Variación Genética/genética , Glicosilación , Células HEK293 , Humanos , Modelos Moleculares , Polisacáridos/química , Polisacáridos/metabolismo , Sialiltransferasas/química , Sialiltransferasas/genética , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Therapeutic performance of recombinant antibodies relies on two independent mechanisms: antigen recognition and Fc-mediated antibody effector functions. Interaction of Fc-fragment with different FcR triggers antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and determines longevity of the antibody in serum. In context of therapeutic antibodies FcγRs play the most important role. It has been demonstrated that the Fc-attached sugar moiety is essential for IgG effector functionality, dictates its affinity to individual FcγRs and determines binding to different receptor classes: activating or inhibitory. In this study, we systematically analyze effector functions of monoclonal IgG1 and its eight enzymatically engineered glycosylation variants. The analysis of interaction of glycovariants with FcRs was performed for single, as well as for antigen-bound antibodies and IgGs in a form of immune complex. In addition to functional properties we addressed impact of glycosylation on the structural properties of the tested glycovariants. We demonstrate a clear impact of glycosylation pattern on antibody stability and interaction with different FcγRs. Consistent with previous reports, deglycosylated antibodies failed to bind all Fcγ-receptors, with the exception of high affinity FcγRI. The FcγRII and FcγRIIIa binding activity of IgG1 was observed to depend on the galactosylation level, and hypergalactosylated antibodies demonstrated increased receptor interaction. Sialylation did not decrease the FcγR binding of the tested IgGs; in contrast, sialylation of antibodies improved binding to FcγRIIa and IIb. We demonstrate that glycosylation influences to some extent IgG1 interaction with FcRn. However, independent of glycosylation pattern the interaction of IgG1 with a soluble monomeric target surprisingly resulted in an impaired receptor binding. Here, we demonstrate, that immune complexes (IC), induced by multimeric ligand, compensated for the decreased affinity of target bound antibody towards FcRs, showing the importance of the IC-formation for the FcR- mediated effector functions.
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Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Secuencia de Carbohidratos , Línea Celular , Cromatografía de Afinidad , Galactosa/inmunología , Galactosa/metabolismo , Expresión Génica , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Datos de Secuencia Molecular , Unión Proteica , Ingeniería de Proteínas , Estabilidad Proteica , Receptores de IgG/genética , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Ácidos Siálicos/inmunología , Ácidos Siálicos/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.
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Citotoxicidad Celular Dependiente de Anticuerpos , Inmunoglobulina G/metabolismo , Ingeniería de Proteínas , Receptores de IgG/metabolismo , Sitios de Unión , Receptores ErbB/genética , Receptores ErbB/metabolismo , Galactosa/metabolismo , Expresión Génica , Glicosilación , Células HEK293 , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Unión Proteica , Receptores de IgG/química , Receptores de IgG/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
BACKGROUND: α-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This "capping" of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins. RESULTS: Human α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates. CONCLUSIONS: Functional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 - 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.