Question 1

Can I use PBS 1X instead of the IgGZERO reaction buffer?

Felicia Mejare · Accepted Answer

Yes, the IgGZERO enzyme is 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.

Question 2

I read that cleavage buffer for IgGZERO is 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 Can I use PBS 1X in place of cleavage buffer?

kk · Accepted Answer

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

Question 3

I did not get complete deglycosylation using IgGZERO Immobilized, how can I improve digestion?

kk · Accepted Answer

First you could increase incubation time and increase temperature to 37°C. One explanation for incomplete deglycosylation with IgGZERO Immobilized 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.

Question 4

What is the difference between GlycINATOR Immobilized and IgGZERO Immobilized?

Felicia Mejare · Accepted Answer

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. IgGZERO Immobilized contains IgGZERO (EndoS) and GlycINATOR Immobilized 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.

Question 5

Which should I choose, GlycINATOR or IgGZERO?

Felicia Mejare · Accepted Answer

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.

Question 6

Which should I choose, GlycINATOR or IgGZERO?

Felicia Mejare · Accepted Answer

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.

Question 7

Is FabRICATOR stable in the presence of denaturing agents such as guanidine hydrochloride or SDS?

kk · Accepted Answer

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.

Question 8

Does FabRICATOR (IdeS) have protease activity on IgM?

kk · Accepted Answer

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.

Question 9

Can I cleave Fc-fusion proteins with FabRICATOR?

kk · Accepted Answer

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.

Question 10

Can I use FabRICATOR and PNGaseF in the same reaction?

Felicia Mejare · Accepted Answer

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.

Question 11

I have a small modification in the sequence below the hinge, will FabRICATOR still digest my IgG?

kk · Accepted Answer

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.

Question 12

I have a small modification in the sequence below the hinge, will FabRICATOR still digest my IgG?

kk · Accepted Answer

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.

Question 13

Is it possible to purchase PNGaseF Immobilized in bulk?

Hanna Toftevall · Accepted Answer

Yes! Contact us at info@genovis.com

Question 14

Is it possible the reuse the  PNGAse F Immobilized column?

Hanna Toftevall · Accepted Answer

No, it is not recommended. We can only guarantee optimal performance for one-time use.

Question 15

Which buffers can be used with the PNGaseF Immobilized column?

Hanna Toftevall · Accepted Answer

The recommended buffer for best performance of the column is TBS buffer at pH 8.6. Other buffers with neutral pH containing 0.15 M NaCl can also be used but optimization may then be required.

Question 16

Can the deglycosylation on the column be performed under denaturing conditions?

Hanna Toftevall · Accepted Answer

The columns are recommended to be used for deglycosylation at native conditions only.

Question 17

Does PNGase F  Immobilized remove all N-linked glycans?

Hanna Toftevall · Accepted Answer

PNGase F Immobilized hydrolyzes the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. It does not remove N-glycans with α1-3 core fucosylation.

Question 18

Does PNGase F  Immobilized remove all N-linked glycans?

Hanna Toftevall · Accepted Answer

PNGase F Immobilized hydrolyzes the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. It does not remove N-glycans with α1-3 core fucosylation.

Question 19

Can I re-use the GlycINATOR Immobilized column?

Felicia Mejare · Accepted Answer

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.

Question 20

What is the difference between GlycINATOR Immobilized and IgGZERO Immobilized?

Felicia Mejare · Accepted Answer

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. IgGZERO Immobilized contains IgGZERO (EndoS) and GlycINATOR Immobilized 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.

Question 21

Which should I choose, GlycINATOR or IgGZERO?

Felicia Mejare · Accepted Answer

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.

Question 22

What is the difference between GlycINATOR and IgGZERO?

Felicia Mejare · Accepted Answer

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.

Question 23

Can I achieve Fc-specific deglycosylation on IgG with N-linked glycans in the Fab region?

Felicia Mejare · Accepted Answer

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

Question 24

Can I achieve Fc-specific deglycosylation on IgG with N-linked glycans in the Fab region?

Felicia Mejare · Accepted Answer

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

Question 25

Can I re-use FabALACTICA Immobilized columns?

Felicia Mejare · Accepted Answer

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.

Question 26

Can I digest Fc-fusion proteins using FabALACTICA (IgdE)?

Felicia Mejare · Accepted Answer

No, FabALACTICA has shown very poor activity on Fc-fusion proteins.

Question 27

Can I release Fc fragments from the CaptureSelect column after isolating the Fab fragments?

Felicia Mejare · Accepted Answer

It is not recommended to elute the Fc fragments from the CaptureSelect column since the fraction might also include undigested or semi digested material.

Question 28

How can I remove residual enzyme from the digested antibody in solution?

Felicia Mejare · Accepted Answer

We recommend using FabALACTICA Immobilized 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.

Question 29

We want to make Fab's directly from antibodies. When would the selection of FabALACTICA be the first choice over GingisKHAN or FabULOUS?

kk · Accepted Answer

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

Question 30

We want to make Fab's directly from antibodies. When would the selection of FabALACTICA be the first choice over GingisKHAN or FabULOUS?

kk · Accepted Answer

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

Question 31

Can I use SialEXO in combination with OglyZOR or OpeRATOR?

kk · Accepted Answer

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

Question 32

Does SialEXO contain a purification tag?

kk · Accepted Answer

Yes, SialEXO contains a 6x His-tag.

Question 33

Can I use SialEXO for desialylation of both N- and O-glycans?

kk · Accepted Answer

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

Question 34

Does SialEXO require denaturation of the proteins for activity on glycoproteins?

kk · Accepted Answer

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

Question 35

Can I use SialEXO in combination with PNGaseF?

kk · Accepted Answer

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.

Question 36

Can I use SialEXO in combination with PNGaseF?

kk · Accepted Answer

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.

Question 37

What enzyme should be selected for the digestion of IgA1?

Maria Ekemohn · Accepted Answer

IgASAP Sub1 specifically targets IgA1 and exhibits a faster reaction time (1 hour) compared to IgASAP Sub1+2 (16-18 hours). The cleavage site of IgASAP Sub1+2 is located six amino acids above that of IgASAP Sub1, resulting in the absence of potentially O-glycosylated residues on the Fab fragments. This characteristic can be advantageous in certain cases.

Question 38

Is there an enzyme for digestion of IgA2-only?

Maria Ekemohn · Accepted Answer

There is no known enzyme that targets IgA2-only since the amino acids in the hinge regions of IgA2 is present also in IgA1. However, IgASAP Sub1 will digest IgA1-only.

Question 39

Can the IgASAP Sub1 digestion be performed at lower temperatures?

Maria Ekemohn · Accepted Answer

The optimal activity is at 37°C, but the digestion can also be performed at room temperature using a prolonged incubation time.

Question 40

Which buffers are compatible with IgASAP Sub1?

Maria Ekemohn · Accepted Answer

The enzyme is active at pH 6.5 to 8.0, tolerates up to 300 mM NaCl, and does not require reducing conditions or co-factors for activity. PBS and TBS buffers are both compatible. Optimization may be required if buffers other than the recommended are used.

Question 41

Does IgASAP Sub1 digest human IgA1-only?

Maria Ekemohn · Accepted Answer

IgASAP Sub1 may digest IgA1 from other primate species with similar hinge sequences, but this has not been tested. IgASAP Sub1 does not digest IgA2.

Question 42

Does IgASAP Sub1 digest human IgA1-only?

Maria Ekemohn · Accepted Answer

IgASAP Sub1 may digest IgA1 from other primate species with similar hinge sequences, but this has not been tested. IgASAP Sub1 does not digest IgA2.

Question 43

Are there any known inhibitors of OpeRATOR?

kk · Accepted Answer

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.

Question 44

Will OpeRATOR work on simple hexose or pentose?

kk · Accepted Answer

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

Question 45

Can OpeRATOR and SialEXO be used in the same reaction?

kk · Accepted Answer

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

Question 46

Is removal of sialic acids really needed?

kk · Accepted Answer

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.

Question 47

Does the enzyme require the sample to be in the native fold or would denaturation help?

kk · Accepted Answer

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.

Question 48

Does the enzyme require the sample to be in the native fold or would denaturation help?

kk · Accepted Answer

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.

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IgGZERO

FabRICATOR Lyophilized

FabRICATOR Low Endotoxin

PNGase F

GlycINATOR

FabALACTICA

SialEXO

IgASAP

OpeRATOR