FAQ

Here you can find all FAQ we have on Genovis products. Don't hesitate to contact us if you can't find an answer to your question.

FabRICATOR

Yes, PNGaseF and FabRICATOR can be used in a one-pot reaction as long as the reaction conditions follow the specifications for the FabRICATOR enzyme. It is important to have native reaction conditions for FabRICATOR to remain active.

Yes, for human IgG1 molecules both enzymes can be used in combination to generate a specific hinge peptide. The incubation time is significantly longer for FabALACTICA and it is recommended to first run the FabALACTICA digest and then add FabRICATOR for the last 30 min.

If we assume that the antibody concentration in serum is 15 mg/ml you would have to add 10-15 000 units to a sample of 1 ml serum. In 50 µl serum you would have to add 500 – 750 units of FabRICATOR.

Yes. FabRICATOR is compatible with EDTA. Make sure your pH is within 6-8.

We have tested 4-6 M GuHCl which the enzyme does not withstand without significant decrease of activity. SDS have not been evaluated but 0.1% RapiGest from waters have negative influence on activity. Taken together the enzyme does not well tolerate denaturing agents although we have not tested this in more detailed studies.

No. According to the following reference, FabRICATOR does not digest IgM. Pavel-Rammingen et al., 2002. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.

Yes, FabRICATOR works on hamster IgG using the standard protocol as for human IgG.

FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (…CPAPELLG / GPSVF…) and the C-terminus will regain the carboxyl group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).

In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2.

We have no stability data for storage at -20°C or -70°C. However, we have customer reporting that they successfully stored reconstituted FabRICATOR at -20°C or -70°C for a couple of months. Genovis cannot guarantee the activity of the enzyme, but these storage conditions might be OK depending on the intended application.

Yes, FabRICATOR should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabRICATOR digests human IgG just below the hinge (…CPAPELLG / GPSVF…), leaving the hinge on the fusion-part.

It is possible to use FabRICATOR with low concentrations of IgG. Low concentrations of IgG will lead to decreased reaction rate, so for concentrations below 0.5mg/mL it is therefore recommended to increase the amount of added enzyme and/or increase the incubation time. There is no risk of over-digestion and the incubation time can be prolonged to O/N if necessary.

FabRICATOR is a very specific enzyme and small changes in the sequence in or around the digestion site (…CPAPELLG / GPSVF…) may reduce or completely impair the ability to digest the IgG. It is very difficult to predict the outcome for each specific modification. It might work, but we cannot guarantee it unfortunately, the only way of knowing is to perform a digestion test. Some suggestions that are worth testing if you experience incomplete digestion: Increase the incubation time, increase the amount of added enzyme, use a PBS with greatly reduced salt concentration (5-10 mM instead of 140 mM) and pH 6.5 instead of 7.4.

FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab’)2 fragment and a Fc-fragment.
Human IgG1: HTCPPCPAPELLG / GPSVF
Human IgG2: VECPPCPAPP_VA / GPSVF
Human IgG3: PPCPRCPAPELLG / GPSVF
Human IgG4: PHAHHAQAPEFLG / GPSVF

FabRICATOR-HPLC

Yes, it is possible to digest the antibody in the first dimension and then separate the subunits in the second dimension. Please see the application note for detailed instructions.

We have tested concentrations as low 0.025 mg/ml without any measurable effect on digestion efficiency. However, low concentration samples require a longer injection time and consequently lead to a broader elution peak from the FabRICATOR-HPLC column.

The FabRICATOR-HPLC can run >400 samples of 10 ug of IgG.

We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR-HPLC as well. However, the ionic strength of the buffer influences retention of the antibody fragments on the FabRICATOR-HPLC column and a minimum of 100-150 mM salt is necessary for IgG1 subunits to elute as a single, narrow peak. Other IgG subclasses or fusion proteins might need some optimization of buffer composition.

FabRICATOR-HPLC is an HPLC column with the FabRICATOR (IdeS) protease immobilized on a HPLC compatible matrix for automated antibody subunit generation.

FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (…CPAPELLG / GPSVF…) and the C-terminus will regain the carboxyl group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).

In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2.

FragIT & FragIT Kit

FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (…CPAPELLG / GPSVF…) and the C-terminus will regain the carboxyl group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).

In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2.

We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Genovis has chosen CaptureSelect in the FragIT Kit for removal of Fc fragments. CaptureSelect has a very high specific affinity for the Fc. Both protein A and MabSelect Sure (GE) may have affinity for the Fab which may result in a lower recovery of the generated F(ab’)2 fragments and the degree of affinity is antibody dependent. We have many customer reports on this subject, and they usually come back to us for the CaptureSelect columns for the above reason.

FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab’)2 fragment and a Fc-fragment.
Human IgG1: HTCPPCPAPELLG / GPSVF
Human IgG2: VECPPCPAPP_VA / GPSVF
Human IgG3: PPCPRCPAPELLG / GPSVF
Human IgG4: PHAHHAQAPEFLG / GPSVF

GingisKHAN®

FabALACTICA (IgdE) and GingisKHAN (Kgp) are both cysteine proteases that digest IgG1 above the hinge to generate intact Fab and Fc fragments. FabALACTICA is specific for human IgG1 and digests at a specific site (…KSCDKT / HTCPPCP…) with overnight incubation without the need for reducing conditions. GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at a specific site (…KSCDK/THTCPPCP…).

The enzyme is a cysteine protease that can be inhibited by alkylating agents such as iodoacetamide. There are also several other known inhibitors to this enzyme, like KYT-36.

We recommend using the 10x reducing agent provided with GingisKHAN which has an optimized pH to provide full enzymatic activity without affecting the disulphide bridges between the light chain and Fd region of the heavy chain, keeping the Fab intact. If other reducing agent is used there is a risk for non-optimal reaction conditions. It is important to use freshly prepared Reducing Agent (max storage 6h) for guaranteed activity of GinigsKHAN.

No, GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at one specific site (…KSCDK/THTCPPCP…) above the hinge, generating Fab and Fc.

Genovis provides a GingisKHAN Fab kit containing a specific Capture Select CH1 resin for further purification of the Fab. See this page for more information.

GingisREX®

No, GingisREX is inhibited by GdHCl even in low concentrations. This chaotrope should be avoided.

The digestion time is affected by many factors like the enzyme to sample ratio, pH of the digestion reaction, and the sample characteristics. The reaction time needs to be optimized for each specific case.

Yes, cysteine is required for both activity and specificity of the GingisREX enzyme.

IgGZERO & deGlycIT

Yes, IgGZERO/deGlycIT are compatible with most commonly used buffers with pH ranging from 6.0 to 8.0, but the reaction conditions might need to be optimized to ensure optimal deglycosylation.

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. The deGlycIT contains immobilized IgGZERO (EndoS) and Immobilized GlycINATOR contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.

IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Typically, GlycINATOR is the first choice since it displays enzymatic activity on all glycoforms present on IgG, including complex type, high mannose, bisected and hybrid type glycans. IgGZERO does not hydrolyze high mannose glycans. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Yes, many buffers are compatible with IgGZERO/DeGlycIT. Please keep pH close to neutral.

First you could increase incubation time and increase temperature to 37°C. One explanation for incomplete deglycosylation with deGlycIT could be that the antibody has higher amounts of high mannose, hybrid glycans or bisected glycans. For these glycans IgGZERO has low or no activity. High mannose glycans are particularly resistant to digestion recommend you try our other endoglycosidase GlycINATOR (EndoS2). This enzyme has much higher activity on these types of glycans sometimes seen in mAbs.

OglyZOR®

No, OglyZOR recognizes the core 1 disaccharide HexNAcHex (or GalNAcGal) and to some extend also the core 3 disaccharide HexNAc(2) (or GalNAcGlcNAc).

Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.

Yes, we have performed successful experiments with OglyZOR, SialEXO and PNGaseF in the same reaction under native conditions O/N at pH ~7.0.  If denaturing reaction conditions are required for PNGaseF, the reaction with OglyZOR and SialEXO needs to be performed first under native conditions, then add PNGaseF and the required buffer.

OglyZOR only works on desialylated O-glycans therefore is a sialidase (SialEXO) included with the product. SialEXO is not O-glycan specific and will therefore desialylate any N-glycans that are present.

OpeRATOR®

OpeRATOR is a metalloprotease and as such highly sensitive to chelating agents such as EDTA. Concentrations >5mM lead to complete inhibition of the enzyme. OpeRATOR activity is also moderately inhibited by ZnCl2.

Yes, the enzymes work in combination and can be used to desialylate and digest an O-glycosylated protein.

No, the enzyme recognizes mucin type glycosylation with an inner GalNAc linked to a hexose sugar.

If digestion is insufficient, it could be caused by the O-glycans having a sterically inaccessible location on the sample that the enzyme cannot reach. In such cases, we recommend trying the following workflow: reduction, denaturation, carboxymethylation, rebuffering, and then digestion with SialEXO and OpeRATOR.

The activity of OpeRATOR is significantly decreased in the presence of sialic acids. We recommend using SialEXO to remove the sialic acids. If this is not an option, then we suggest trying to digest both with and without SialEXO to evaluate the impact for your specific samples and requirements.

The relative abundance and site occupancy of O-glycosylation may vary a lot in a glycoprotein sample, and this will result in the generation of peptides with variable sizes. In some cases, the peptides contain more than one O-glycan, i.e. OpeRATOR does not digest at every site in every molecule. However, the missed digestion sites differ from molecule to molecule, and the compiled data will provide valuable information on the O-glycosylated sites.

Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.

SialEXO®

Yes, SialEXO can be incubated together with OpeRATOR and/or OglyZOR in 20 mM Tris pH 6.8.

Yes, you can. All three enzymes perform well in the same reaction buffer (20 mM Tris pH 6.8). There is no need to increase incubation time when combining the enzymes.

Yes, SialEXO contains a 6x His-tag.

Yes, both enzymes are active under native conditions, preferably use 20 mM Tris pH 7. If denaturing reaction conditions are required for PNGaseF, the reaction with SialEXO needs to be performed first under native conditions, then add PNGaseF and the required buffer.

No, SialEXO works both on glycoproteins in native conditions but can also be used on denatured proteins after buffer exchange.

If you have a technical question, please submit a support case.

Yes, SialEXO displays activity on sialic acids with α2-3, α2-6 and α2-8 linked bonds of both N- and O-glycans.

General

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All FAQ

The GlyCLICK technology can be used to conjugate IgG with Desferrioxamine (DFO) using the GlyCLICK DFO kit. The DFO is a chelating agent for radiolabeling with the radioisotope Zirconium-89 (89Zr) for PET-imaging. The Azide Activation kit can be used to conjugate a chelator of choice if it is alkyne-modified (carrying for example DIBO, DBCO or BCN) to be compatible with the click chemistry.

GlyCLICK is available in kit formats with AlexaFluor®488, AlexaFluor®555, AlexaFluor®647 or in the Azide Activation kit format for conjugation of a label of choice. With the fluorescent label being alkyne-modified (carrying for example DIBO, DBCO or BCN) it is possible to combine it with the GlyCLICK azide activation kit.

The stability of azide activated material varies depending on the antibody and if stabilizing agents have been added. In our labs we have stored azide activated antibodies in TBS buffer without any additives at +4°C for over two years. The azide activation was still OK, and the click reaction generated antibodies with DOL=2. If azide is added as a preservative during long-term storage, it must be removed prior to click reaction.

The final step of GlyCLICK labeling kit is based on a copper free click reaction (SPAAC) and it is a strain promoted reaction between an azide and an alkyne-carrying label. As the name indicates the reaction occurs spontaneously due to the strained bond angles. The use of this click reaction that forms stable conjugates, allows you to freely choose label or payload to combine with your IgG as long as it is alkyne-functionalized. Several alkyne-carrying labels are commercially available with cyclooctyne structures such as DIBO, DBCO or BCN.

Yes, FabULOUS should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabULOUS digests human IgG1 just above the hinge (…KTHT / CPPCP…).

All the steps (deglycosylation, azide activation and click reaction) in GlyCLICK are highly efficient. Normally, all molecules are conjugated when using GlyCLICK, resulting in DOL=2. On the application page we have shown by MS-analysis that all molecules have been conjugated and there is no remaining un-conjugated material.

FragIT digests rabbit IgG but to capture the Fc fragments in the affinity step you need another resin than the Capture Select Fc normally included in the kit. Please contact the Genovis team if you are interested in processing rabbit IgG and we will provide columns with resin compatible with rabbit Fc in the FragIT kit.

FabULOUS is a cysteine protease that digests IgG in the hinge region. A primary digestion site for human IgG1 is above the hinge (…KTHT / CPPCP…). The enzyme is also active on human IgG2 and IgG3, digestion site not determined but Fab and Fc is generated. FabULOUS digests human IgG4 primarily at a site below the hinge using significantly longer incubation, generating Fab’ due to the reducing conditions. The possibility of other, secondary digestions sites is antibody dependent

Characterization using LC-MS will give a complete and clear picture of mass shift associated with successful conjugation if the antibody is analyzed reduced or fragmented. LC or LC-MS analysis of HC from a reduced sample or scFc fragments generated by FabRICATOR digestion provides distinct elution peaks for conjugated scFc or mass shifts corresponding to the degree of labelling (DOL) following conjugation. On the application page you can see an example of such analysis. Analysis of fluorescently labeled antibodies can also be performed by absorbance measurements and calculate the DOL using the Extinction coefficient for the antibody and the label and the correction factor for the label.

The GlyCLICK 250 ug kit is optimized for 250 ug of IgG as a starting material. We do not recommend using less than 100 ug of starting material in a minimum of 100µL to achieve proper end-over-end mixing on the GlycINATOR columns. Please contact support@genovis.com for assistance if you want to process smaller amounts then 100µg.

No, GlycOCATCH is designed to bind specifically to mucin type (core 1) O-glycosylated proteins and peptides. The performance is significantly enhanced by removal of sialic acids using SialEXO.

Unfortunately, FabRICATOR Z does not digest rat IgG2a. If generating Fab fragments is an option, we suggest using FabULOUS enzyme that digests rat IgG2a. FabULOUS does not have a single specific cleavage site, but a primary digestion site, so there can be more than one digestion site. The digestion site(s) for rat IgG2a using FabUSLOUS is not determined, but the generated fragments are Fab and Fc.

Yes, IgGZERO/deGlycIT are compatible with most commonly used buffers with pH ranging from 6.0 to 8.0, but the reaction conditions might need to be optimized to ensure optimal deglycosylation.

We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Yes. There is the possibility to exchange the CaptureSelect LC-kappa (mur) columns to columns containing CaptureSelect LC-lambda (mouse). This resin is specific for mouse IgG. Please contact support@genovis.com if this is of interest.

Depending on what reducing agent and what concentration you use you can obtain intact Fab and not reduce the interchain disulfide bond. The light chain (LC) and heavy chain (HC) will not dissociate under native conditions due to hydrogen/hydrophobic forces. Before you analyze your samples with SDS-PAGE you must remove the cysteine otherwise the Fab will be reduced to Fd and LC during sample preparation for the SDS-PAGE. This can be done by a simple buffer exchange.

No, FabRICATOR Z only digests mouse IgG2a (….CPAPNLLG / GPSVF.. ) and IgG3 (…CPAPNILG / GPSVF…) at one specific site below the hinge. If Fab fragments are an option, we recommend our FabULOUS enzyme that digest mouse IgG1 above the hinge. FabULOUS requires light reducing conditions for optimal activity. If strong reducing conditions are used, this may likely cause reduction of the interchain disulfides.

We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

FabRICATOR MagIC is suitable for using in automated systems that can handle magnetic beads. On the applications page you can read about how it’s used in a KingFisher™ workflow.

IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

We can only guarantee optimal deglycosylation for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. The deGlycIT contains immobilized IgGZERO (EndoS) and Immobilized GlycINATOR contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.

The protocol for FabRICATOR MagIC is based on using standard PBS pH 7.4 as digestion buffer. FabRICATOR in solution is compatible with most commonly used buffers at pH=6-8 and the same buffers should work for FabRICATOR MagIC, but the incubation time might need to be optimized.

Yes, it is possible to digest the antibody in the first dimension and then separate the subunits in the second dimension. Please see the application note for detailed instructions.

Yes, PNGaseF and FabRICATOR can be used in a one-pot reaction as long as the reaction conditions follow the specifications for the FabRICATOR enzyme. It is important to have native reaction conditions for FabRICATOR to remain active.

We recommend using Immobilized FabALACTICA for digesting human IgG1 and generating a final preparation free of any residual enzyme. You can also use FabALACTICA Fab kit for digestion of human IgG1 and purification of the generated Fab fragments.

Typically, GlycINATOR is the first choice since it displays enzymatic activity on all glycoforms present on IgG, including complex type, high mannose, bisected and hybrid type glycans. IgGZERO does not hydrolyze high mannose glycans. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Yes, for human IgG1 molecules both enzymes can be used in combination to generate a specific hinge peptide. The incubation time is significantly longer for FabALACTICA and it is recommended to first run the FabALACTICA digest and then add FabRICATOR for the last 30 min.

Yes, the GlycINATOR enzyme binds specifically to the Fc region and therefore will not deglycosylate N-linked glycans in the Fab region.

The FabRICATOR MagIC magnetic beads with immobilized FabRICATOR (IdeS) enzyme.

FabALACTICA (IgdE) and GingisKHAN (Kgp) are both cysteine proteases that digest IgG1 above the hinge to generate intact Fab and Fc fragments. FabALACTICA is specific for human IgG1 and digests at a specific site (…KSCDKT / HTCPPCP…) with overnight incubation without the need for reducing conditions. GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at a specific site (…KSCDK/THTCPPCP…).

Yes. IMPORTANT! Before you load your sample make sure that the protease in solution is inactivated and that the pH, buffer, chaotrope and NaCl concentrations are within the recommended ranges according to the instructions.

We have tested concentrations as low 0.025 mg/ml without any measurable effect on digestion efficiency. However, low concentration samples require a longer injection time and consequently lead to a broader elution peak from the FabRICATOR-HPLC column.

The OpeRATOR enzyme digests O-glycosylated proteins N-terminally to the serine (S) and threonine (T) glycosylation sites. OpeRATOR therefore facilitates mapping of O-glycosylation sites by LC-MS and as the digested products no longer bind to the GlycOCATCH resin, elution and sample preparation can be achieved in a single step. However, this requires the O-glycosylation sites to be spaced closely together for the OpeRATOR digestion to yield peptides of a size suitable for analysis by LC-MS/MS.

No, when the glycoprotein is digested with OpeRATOR, the binding capacity of GlycOCATCH is lost.

No. Branching fucose linked to the same residue as the β-galactose inhibits GalactEXO activity and needs to be removed prior to GalactEXO treatment. The FucosEXO enzyme can be used for hydrolysis of α1-2, α1-3 and α1-4 linked fucose.

No, FabULOUS Fab kit contains CaptureSelect LC-kappa (mur) spin columns with selective affinity for murine LC-kappa. If you need to generate pure Fab from human IgG1 you can use either FabALACTICA Fab Kit or GingisKHAN Fab Kit. If you have a different subclass of human IgG, please contact support@genovis.com for guidance.

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No, GingisREX is inhibited by GdHCl even in low concentrations. This chaotrope should be avoided.

Unfortunately no, the enzyme requires trimming of the Fc-glycan using GlycINATOR to enable access to the core fucose substrate.

Yes, the transglycosylation reaction can be performed on all subclasses of human IgG. The reaction is somewhat slower on IgG2 and longer incubation times may be necessary to obtain over 95% transglycosylation.

No, TransGLYCIT is based on IgG specific enzymes and will only transglycosylate IgG.

The FabRICATOR-HPLC can run >400 samples of 10 ug of IgG.

We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR-HPLC as well. However, the ionic strength of the buffer influences retention of the antibody fragments on the FabRICATOR-HPLC column and a minimum of 100-150 mM salt is necessary for IgG1 subunits to elute as a single, narrow peak. Other IgG subclasses or fusion proteins might need some optimization of buffer composition.

GlycINATOR is an IgG specific endoglycosidase that hydrolyzes complex, hybrid and high mannonse type glycans on the conserved Fc site on IgG.

Yes, many buffers are compatible with IgGZERO/DeGlycIT. Please keep pH close to neutral.

The TransGLYCIT platform is developed for transglycosylation of human IgG.

The digestion time is affected by many factors like the enzyme to sample ratio, pH of the digestion reaction, and the sample characteristics. The reaction time needs to be optimized for each specific case.

FabRICATOR-HPLC is an HPLC column with the FabRICATOR (IdeS) protease immobilized on a HPLC compatible matrix for automated antibody subunit generation.

OpeRATOR is a metalloprotease and as such highly sensitive to chelating agents such as EDTA. Concentrations >5mM lead to complete inhibition of the enzyme. OpeRATOR activity is also moderately inhibited by ZnCl2.

First you could increase incubation time and increase temperature to 37°C. One explanation for incomplete deglycosylation with deGlycIT could be that the antibody has higher amounts of high mannose, hybrid glycans or bisected glycans. For these glycans IgGZERO has low or no activity. High mannose glycans are particularly resistant to digestion recommend you try our other endoglycosidase GlycINATOR (EndoS2). This enzyme has much higher activity on these types of glycans sometimes seen in mAbs.

The enzyme is a cysteine protease that can be inhibited by alkylating agents such as iodoacetamide. There are also several other known inhibitors to this enzyme, like KYT-36.

Yes, both beta1-3 linked galactose on O-glycans and beta1-4 galactose on N-glycans are hydrolyzed by GalactEXO.

Yes, SialEXO can be incubated together with OpeRATOR and/or OglyZOR in 20 mM Tris pH 6.8.

We recommend using the 10x reducing agent provided with GingisKHAN which has an optimized pH to provide full enzymatic activity without affecting the disulphide bridges between the light chain and Fd region of the heavy chain, keeping the Fab intact. If other reducing agent is used there is a risk for non-optimal reaction conditions. It is important to use freshly prepared Reducing Agent (max storage 6h) for guaranteed activity of GinigsKHAN.

For most substrates such as antibodies or Fc-fusion proteins a 2 h incubation results in hydrolysis of over 95% of the present galactose. More complex substrates with high degree of galactosylation may require longer incubation times.

No, GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at one specific site (…KSCDK/THTCPPCP…) above the hinge, generating Fab and Fc.

Yes, cysteine is required for both activity and specificity of the GingisREX enzyme.

Genovis provides a GingisKHAN Fab kit containing a specific Capture Select CH1 resin for further purification of the Fab. See this page for more information.

Yes, you can. All three enzymes perform well in the same reaction buffer (20 mM Tris pH 6.8). There is no need to increase incubation time when combining the enzymes.

Yes, SialEXO contains a 6x His-tag.

Yes, both enzymes are active under native conditions, preferably use 20 mM Tris pH 7. If denaturing reaction conditions are required for PNGaseF, the reaction with SialEXO needs to be performed first under native conditions, then add PNGaseF and the required buffer.

No, SialEXO works both on glycoproteins in native conditions but can also be used on denatured proteins after buffer exchange.

Yes, SialEXO displays activity on sialic acids with α2-3, α2-6 and α2-8 linked bonds of both N- and O-glycans.

Yes, the enzymes work in combination and can be used to desialylate and digest an O-glycosylated protein.

Yes. FabRICATOR is compatible with EDTA. Make sure your pH is within 6-8.

If we assume that the antibody concentration in serum is 15 mg/ml you would have to add 10-15 000 units to a sample of 1 ml serum. In 50 µl serum you would have to add 500 – 750 units of FabRICATOR.

No. According to the following reference, FabRICATOR does not digest IgM. Pavel-Rammingen et al., 2002. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.

We have tested 4-6 M GuHCl which the enzyme does not withstand without significant decrease of activity. SDS have not been evaluated but 0.1% RapiGest from waters have negative influence on activity. Taken together the enzyme does not well tolerate denaturing agents although we have not tested this in more detailed studies.

Yes, FabRICATOR works on hamster IgG using the standard protocol as for human IgG.

FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (…CPAPELLG / GPSVF…) and the C-terminus will regain the carboxyl group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).

In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2.

No, the enzyme recognizes mucin type glycosylation with an inner GalNAc linked to a hexose sugar.

If digestion is insufficient, it could be caused by the O-glycans having a sterically inaccessible location on the sample that the enzyme cannot reach. In such cases, we recommend trying the following workflow: reduction, denaturation, carboxymethylation, rebuffering, and then digestion with SialEXO and OpeRATOR.

The activity of OpeRATOR is significantly decreased in the presence of sialic acids. We recommend using SialEXO to remove the sialic acids. If this is not an option, then we suggest trying to digest both with and without SialEXO to evaluate the impact for your specific samples and requirements.

The relative abundance and site occupancy of O-glycosylation may vary a lot in a glycoprotein sample, and this will result in the generation of peptides with variable sizes. In some cases, the peptides contain more than one O-glycan, i.e. OpeRATOR does not digest at every site in every molecule. However, the missed digestion sites differ from molecule to molecule, and the compiled data will provide valuable information on the O-glycosylated sites.

No, OglyZOR recognizes the core 1 disaccharide HexNAcHex (or GalNAcGal) and to some extend also the core 3 disaccharide HexNAc(2) (or GalNAcGlcNAc).

We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.

It is possible to elute the Fc fragments from the CaptureSelect column using a low pH buffer, for example a 0.1M Glycine buffer pH=3.0. The pH must immediately after elution be adjusted to neutral pH by adding 0.1x elution volume of 1M Tris pH=8.0.

Yes, we have performed successful experiments with OglyZOR, SialEXO and PNGaseF in the same reaction under native conditions O/N at pH ~7.0.  If denaturing reaction conditions are required for PNGaseF, the reaction with OglyZOR and SialEXO needs to be performed first under native conditions, then add PNGaseF and the required buffer.

OglyZOR only works on desialylated O-glycans therefore is a sialidase (SialEXO) included with the product. SialEXO is not O-glycan specific and will therefore desialylate any N-glycans that are present.

Yes, but a longer incubation time is needed.

Yes, FabULOUS requires reduced condition for activity and reaction is performed in the presence of 1-5 mM DTT or TCEP at neutral pH.

FabULOUS enzyme reaction is inhibited with iodoacetamide, iodo acetic acid or lowering pH below 3.

FabALACTICA does not require reducing conditions as both GingisKHAN and FabULOUS does, and allows for generation of Fab fragments from native human IgG1.

We have no stability data for storage at -20°C or -70°C. However, we have customer reporting that they successfully stored reconstituted FabRICATOR at -20°C or -70°C for a couple of months. Genovis cannot guarantee the activity of the enzyme, but these storage conditions might be OK depending on the intended application.

Genovis has chosen CaptureSelect in the FragIT Kit for removal of Fc fragments. CaptureSelect has a very high specific affinity for the Fc. Both protein A and MabSelect Sure (GE) may have affinity for the Fab which may result in a lower recovery of the generated F(ab’)2 fragments and the degree of affinity is antibody dependent. We have many customer reports on this subject, and they usually come back to us for the CaptureSelect columns for the above reason.

Yes, FabRICATOR should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabRICATOR digests human IgG just below the hinge (…CPAPELLG / GPSVF…), leaving the hinge on the fusion-part.

It is possible to use FabRICATOR with low concentrations of IgG. Low concentrations of IgG will lead to decreased reaction rate, so for concentrations below 0.5mg/mL it is therefore recommended to increase the amount of added enzyme and/or increase the incubation time. There is no risk of over-digestion and the incubation time can be prolonged to O/N if necessary.

FabRICATOR is a very specific enzyme and small changes in the sequence in or around the digestion site (…CPAPELLG / GPSVF…) may reduce or completely impair the ability to digest the IgG. It is very difficult to predict the outcome for each specific modification. It might work, but we cannot guarantee it unfortunately, the only way of knowing is to perform a digestion test. Some suggestions that are worth testing if you experience incomplete digestion: Increase the incubation time, increase the amount of added enzyme, use a PBS with greatly reduced salt concentration (5-10 mM instead of 140 mM) and pH 6.5 instead of 7.4.

FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab’)2 fragment and a Fc-fragment.
Human IgG1: HTCPPCPAPELLG / GPSVF
Human IgG2: VECPPCPAPP_VA / GPSVF
Human IgG3: PPCPRCPAPELLG / GPSVF
Human IgG4: PHAHHAQAPEFLG / GPSVF

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