Dr. Atis Chakrabarti: Good morning, everybody. It's very nice to meet you here. And it's my pleasure to give this talk on our very popular SEC column, particularly GFC aqueous SEC column for the separation of the biomolecules

And as you see from the title of the talk, I'll just give you a brief overview of the columns available for the separation of the biomolecules. And I'll touch base upon the common issues which you find all the time during method development and troubleshooting

Next slide, please. Next slide shows the overview, how the talk will go. So, I'll introduce about the TSKgel different types of columns available. Then what are the factors when we do a chromatographic analysis? And I found this particular one in an LC-GC article. I'll just talk about it because there's a very nice guideline for taking care of the column

And slide seven, basically, when you start SEC, you expect some features a column should have and how we can maintain those features in the column. That's the--particularly for the protein separation and peptide separation

And slide nine is the overview of the column line, SW column line, which is basically the most popular column line for the separation of the proteins and the application of the monoclonal antibody and so on

And the slides nine to 14 will cover the column selection guide for the biomolecules in--little bit in broader perspective

And then applications, I'll select mostly the applications which is based on the separation of the proteins, monoclonal antibodies, peptides, and so on, separation of the monomer from the dimer or aggregates like that

And as you see here, I put the column lifetime in red because that's very important issue. When you spend some $1,700 or so, you need a column which is very stable. So, I'll talk about that little more in details

And slide 37 to 41, it's about the secondary interaction because, as you know, the size exclusion chromatography is the only mode of separation where you don't need any interaction

So--but, in the ideal world, it is not possible. There are some situations when you'll have interaction, so how far we can manage that and definitely the conclusion

This is just one slide to show that we have a broad range of the particle size available through our inventory. And some of them are in pre-packed column form. And some of them are bulk media. Some of them you can actually get in both bulk media and pre-packed column form

We have from 2 micrometer to 100 micrometer, that wide range of particle size. So, we have a scope of seamless actually scale up and scale down as you require

This is a puzzle. Anybody, when you are trying to solve the chromatographic conditions based suitable for your separation, you have to solve this puzzle. And that involves ligand, pH, salt type, buffer concentration, temperature, gradient, column dimension, particle

Now, it depends on what mode you are using. But, even in SEC, there are so many factors in this puzzle which we have to solve before you can actually get a clear-cut chromatographic separation method

Next slide, please. Now, I have listed some of the factors which actually affect the separation for the protein and peptides. Complex mixture is important problem. Low sample concentration is another factor

Sometimes, the sample is very costly. You don't want to waste. So, you need some column which is very highly sensitive and can separate. And now it is LCMS [sp] type of application is becoming very important. So, we should have some column which is suitable for that type of application

Aggregation is an issue. Aggregation, sometimes inherent aggregation, sometimes it's not inherent. So, these are all--maybe because of the secondary interaction also, and when we develop the methods, particularly the chromatographic conditions, we have to take care of all these things, types of salt, what is the salt concentration, because we have a very nice database in our inventory where we have developed lot of applications where we have shown that the salt concentration plus pH, all these actually affect the overall chromatographic separation

Surfactants, surfactants are--many times is needed for the protein separation at some point. And even surfactant is needed sometimes to increase in the separation and better separation for the molecules also

Other derivatives--additives, denaturant, and heat, everything--heat is not that much important in SEC. But, other things are very important when you are solving that puzzle for the best mode of chromatographic separation

Next slide, please. Now, I got this particular one from LC-GC. You know John Dolan? He's very famous. He writes so many different articles. And one was 10 commandments for the column lifetime

And definitely, all of these are very important. And we try to take care of all these as much as possible. And that helps in increasing the column lifetime. So, dedicate the column, particularly dedicate the column is more important when you are using some kind of additives

Use in-line filters, flush column regularly, guard columns, we always encourage the end users and scientists that please use the guard column because the guard column actually helps in trapping all those dirty materials from entering into the column

And whether your column is now under pressure from the dirty materials or not, that you can actually figure it out just by change in the pressure in the back pressure in the system

Avoid the void, control the temperature, retire early, I mean, he's saying that sometimes we think that columns should be used forever. But, at some point, you have to think that, no, it's time to retire

Ensure the supply, minimum garbage, and purity is good

So, we have--actually, if you go through the blog, we have in our Website tosohtalk.com, there is a very nice blog how long the column last. And I would request you whenever you have time, just go through the blog. It's a very nice blog, detailed discussion about the pros and cons of the column lifetime is discussed in that particular blog

Next slide, please. Now, when you buy a column, what you expect from that column, there are certain features, like rigid solid support, rigid silica support, which we have. We have tested the mechanical strength of our silica beds

High resolution and sharp peak, fast separation, reproducibility, column stability, analytical and preparative size column, which we have, high recovery, and I'll show you some slides where we have shown that we have a very high recovery of all the SEC columns or GFC columns we have for the separation of the biomolecules

And the basic idea of using SEC, particularly GFC, is to maintain the biological activity. And so, these are the main features we expect from the SEC column, GFC column for the separation of the protein

Now, in the field of protein and peptide analysis, size exclusion column is basically industry's workhorse. Nowadays, the--particularly in the monoclonal antibody separation field, 99.9 percent purity is sometimes desired when we cannot get just by one chromatographic more

But, at the last phase, we use the size exclusion chromatography for the separation and quality control of the protein we separate, monoclonal antibody we separate

So, another factor, just like aggregation analysis is also very important. And GFC has a very important role in separating the monomer from the dimer, trimer, and so on

This is just a tableted form of the different types of columns we have from our inventory. And they're parameters, like particle size, pore size, different types of standards we have used to calibrate that particular column

We have used different standards because all proteins are not globular. So, some proteins are not globular. So, we have used globular, dextrans, globular protein, dextran [unintelligible] PEG, linear, branched, and globular

So, depending on the situation, depending on the protein interest, interest of your substance, you have to select which calibration car [sp] you have to choose. And we have the different molecular weight, range available for each of these columns. And you can select using this table the best column for your separation

Now, couple of things--no, go back, please. Yeah, couple of things, I'll talk about the pore size, for example. As you go down from 2000 to 4000, as you see, the pore size changes from low to high, 125, 250, and 450

Now, you have seen here there is a column called BioAssist column in this particular one. As you know, the SEC column, when comes in steel housing material, there is a situation where you can get the interaction from the housing material itself

And if you suspect that you are getting that type of interaction from housing material, I would request you to go to the BioAssist column. They are basically same column, just like stainless steel column, only in the peak hardware

Now, if you look at the SWXL and SuperSW column line, you see we have 125, 250, 450 and here also 125, 250, and 450, three different pore sizes. And in this case, we have two different pore sizes for super

Next slide, please. So, as I said that the SEC basic principle of SEC is that the column will separate just on the basis of hydrodynamic radius of the molecule. Now, that is the molecule receiving mechanism, which you have to somehow in the hands to get the best separation as a function of size. But, that's not the ideal situation

So, what happens to prevent any kind of a secondary interaction? The column is protected by a diol coating, which is kind of proprietary diol coating to shield the protein surface from coming into contact with the--I mean, column surface from coming into contact with the protein surface and any interaction

Next, please. Now, as I have shown you that, irrespective of SW, SWXL, we have three different pore sizes, and for SuperSW only, we have two different pore sizes. We have found that the G3000 column line is the best column line for the most protein sample separation and the monoclonal antibody separation

Next, please. Now, what is the difference between the SEC columns we have compared to the SEC columns you get from the other vendors? The--our column has the more pore volume per unit column volume, which results into the better selectivity and resolution

And as you go from the SW to SuperSW, so we have a column line, SW column line. Within that particular column line, we have SW, SWXL, and SuperSW. The difference is the particle size. Particle size decreases from SW to SuperSW

Now, as you go down the particle size, you know that pressure will increase very rapidly. Now, this SuperSW generally is used for LCMS [sp] type of application or if you have a very dilute sample or you have a very costly sample you don't want to waste at all in those cases because it has higher sensitivity compared to the other columns

Next, please. Now, this is just one calibration curve. I mean, I'm showing here we have lots of different calibration curves which you can use

As you see, we have used the linear standard, which is PEG, then branch dextran and protein globular, spherical to draw the calibration curves

So, right now, we'll concentrate on the solid black circle. So, that is the calibration curve for the protein for G2000. And next one, next panel is G3000SW. And next panel is G4000SW column

Now, next, please. If you look at the middle panel, which is G3000SW column, and if you concentrate--if you focus on this particular dot, which represents one 150 kilodalton molecular weight, which is the molecular weight in general for the monoclonal antibody, that is exactly somewhere in the plate or in between the total exclusion and total inclusion

That's why this 3000SW column line is most popular for the monoclonal antibody and protein separation of that molecular weight range

But, if you look at the same molecular weight here, it is almost to the total inclusion, whereas here, it is total exclusion. That's why G3000SW or SWXL is the best column for the separation of the monoclonal antibody or a protein of that particular molecular weight or so

Now, again, this is just a shorter version so that you can compare your standards, linear, branched, and globular as a function of different types of column line I have available for the separation

If you know your molecular weight and if you know your nature of the sample, if you know that it's a--it's having a molecular weight between 2,000 and 70,000, and if you know that this is a branched molecular structure, then you know which column you should use

Next slide, please. Now, for example, you have no idea what column you have to use because I have no idea what the molecular weight of it. So, we generally request that use the G3000SWXL column as a scouting column

We have another column which is 15-centimeter column for this, which is QC-packed GFC, that is even better. And you can use that for scouting, and then you can come back to G3000 also

Now, depending on--previous, please. Depending on the elution time, whether it's coming in the exclusion or it's coming in the total inclusion, you have to select either G4000 or G2000SWXL column

Next slide, please. Now, what is the difference between the XL column line and the SW--SW column line and the XL column line? In terms of efficiency, if you--this is the Y axis where HETP is plotted as a function of the sample load

This is about the XL, and this is about SW. In general, SWXL column line, because of the lower column size, performs much better compared to SW column line. But, in terms of loading capacity, they are more or less same, about 250 micro--we generally say 100 microgram. But, strictly speaking, even 250 microgram works pretty well without much loss in efficiency. But, after that, it starts dropping very, very quickly

Next, please. Now, we have done some recovery study as a function of three different column lines of XL series, 2000, 3000, and 4000. As I'm not going into the fine details, but at a glance, you can clearly see that, even at 100 microgram of the sample load, the overall recovery is very, very good, irrespective of the different types of pore size. Excuse me

We have used the three different types of molecules here. As you see, thyroglobulin in this case, it is the 78, which is low compared to the others. And the research shows that mostly, it's the protein specific, not really column specific. Otherwise, the recovery is very, very quantitative and very high

Next slide, please. So, this is just, again, another slide just to show we have different types of column lines available for different types of applications. And we have a selection criteria depending on what sample, nucleic acid, DNA, RNA, oligonucleotide, proteins, it's a large protein or small protein, what kind of protein

Next slide, please. Then it's a virus or it's a peptide. You can use this table to figure it out, what is useful for you. For virus and very large protein like IgM or so, generally, we recommend 6000 or 4000 or 6000, something like that; 4000 is generally used for IgM, viruses, 6000, 5000, or so

We--as you see, there is a difference here, PW. So, what is the difference between C--SW and PW? SW means--S stands for silica. W means water loving. So, from the nomenclature itself, you can figure it out whether it's a silica-based column or polymer-based column. Mostly, the proteins are separated using silica-based column

Next slide, please. This is a reference I took from an article recently published in this particular journal. It's a very nice article where they have used TSKgel G3000SWXL column for the separation of the IgG monoclonal antibody. And they have actually induced the aggregation as a function of light. And there are so many ways you can actually induce the aggregation

The bottom line of this particular article is that you can use TSKgel G3000SWXL column to separate the monomer from dimer and other--the multimers. And in the--I'm not going into the fine details. But, they have shown that this is more efficient if you can use two wavelengths. As you see, they have done double wavelength SEC here. They have used 240 nanometer [unintelligible] 280 nanometer for this study

But, as you see, depending on the type of aggregation method, they are getting different amounts of aggregate but could be separated very well using this particular column

Now, this is the study we have done in our lab. We have used monoclonal antibody just to see whether the column has any effect on the amount of the monoclonal antibody and loading on the column

So, we started the blue line--I'm not sure if the color is very much clear. But, this is--blue line is 40 microgram of the monoclonal antibody. And the rate is 400 micron. We just aimed for the higher amount of the monoclonal antibody

And we have tabulated the retention time, efficiency, and asymmetric factor as a function of the amount. And as you see, the retention time is 8.023, 8.061, 4500, 3508, and asymmetric factor 1.22, 1.35

The bottom line of this is the peak efficiency as well as the shape did not change dramatically, though we have loaded 10-fold amount of the monoclonal antibody here. This is the zoomed-in figure where you have seen the separation of the fragments and the monomer from the dimer and aggregates

As you know that, in the therapeutic, you know, separation --I mean, therapeutic IgG, you have to report how much albumin protein is there. And so, what we have done here, we have added 20-fold excess of IgG compared to the concentration of the albumin. And we tried to find out whether we can separate that and see that

Actually, as you see, this is the human IgG and can be separated from the 20-fold--I mean, albumin and 20-fold excess of IgG from this particular chromatographic conditions, which is very common chromatographic conditions for the separation of the monoclonal antibody

Next slide, please. Digestion, digestion is another important thing. So, where we have done--we have tracked the codes of the change of the enzyme as a function of the time when you digest. So, we have digested the IgG by pepsin

And as you see, as a function of time, the monomer peak goes down. The other peaks comes up. And they could be very, very nicely separated to the best line. And you can actually monitor as a function of time the amount of the digested products and the loss of the monomer peak

Next slide, peak. PEGylation is another important issue for the--there are so many factors like you need a targeted delivery. You need a toxicity-related issue you want to avoid. So, anyway, we have used three different molecular weights of PEG. And we derivitized [sp] the lysozyme with that. And the red is the position of the six lyshin [sp] residues where it's getting derivitized

And we tried to separate--next slide, please--using the TSKgel SWXL column, 3000SWXL column. And solid line 5KD, next is 10, and next is 30 of the PEG

And as you see, irrespective of the PEG molecular weight, we could separate the mono from the other, mono, di, and the lysozyme peak very nicely. This is the tri-PEG PEGylation

So, that shows that this particular column is very nice for the separation of the different PEGylated proteins

Next slide, please. This is the separation of the peptides using this column, TSKgel G3000SW. In this case, it is not XL. It is SW column. And we have used a number of the different types of peptides. We have added 0.2 percent SDS surfactant in this case. And we could separate all of them very, very well, almost to the baseline

And this is the list of the different peptides we tried. So, this is very nice application to show that we have applications without the SDS also for another set of peptides. And if you are interested, anytime, we can get that from our database

Next slide, please. Now, can we separate the two peptides differing just by one amino acid? We have done that here using SuperSW2000 column. And as you know, the super means it's a 4-micron particle size, smaller particle size

Now, next slide, please. As you see, peptide 20 and peptide 22, they are differing in only one amino acid. If you look into the chromatographic conditions now, this is not exactly same as we did in other cases. Here, we have added 45 percent aqueous acetyl nitrate. So, it is acetyl nitrate--containing acetyl nitrate

And it has 0.1 percent TFA also. And under that chromatographic conditions, actually, you can separate the two peptides just differing by only one amino acid. This is a very nice application to show that type of--it's not hardcore SEC in that sense. It has some other factors, hydrophobic interactions and all these things also. But, it's a very nice application to show that you can do that

Next, please. Now, what is the chaotropic agent's effect on the separation? In this case, we have separated the fusion protein. And we have added sodium perchlorate in the buffer

And as you see, when we used 0.4 moles per liter sodium perchlorate into the buffer, the tailing was improved. The--you have to follow the green line, which is the--green line is the separation using 0.4 moles per liter sodium perchlorate

It definitely improved the tailing. Not only it improved the tailing, it could see more of your aggregate present in that. So, again, it is done using SuperSW 3000 column, which is 4-micron particle size column

Next slide, please. Recently, you have noticed that in LC-GC, there is a very nice article about the column lifetime. And if you notice that, over so many years, 2007, 2009, and 2011, the overall perception of the scientist end users have not changed. They think that column to column reproducibility, column lifetime matter more than the price. And that's understandable

And if you compare the total effect of those two over the price, it is 37 percent against 12, 39 percent against 14, 34 percent against 13 percent. So, it's very important that the end users want a column which is stable, and it's a good column

So, we did some study to see how much is the column lifetime we have in our case. And we have used very simple standard chromatographic conditions first because that's the chromatographic conditions we use for our quality control, 100 millimolar phosphate buffer, pH 6.7, containing the neutral salt, sodium sulfate, and sodium is just to avoid any kind of bacterial growth

And we have used the standard SWXL mixture containing four different standard proteins and one small molecule PABA. And we have used monoclonal antibody in another experiment also

Next, please. Remember, when we talk to our scientists, we always request or we always recommend that please use the guard column. Please filter the buffer and the sample through the 0.45-micron unit. All these things we recommend. But, to give the column extra pressure, we did not use those two conditions in our study as well as we did not clean the column in between at all from the injection number one to injection number 1,000

And these conditions, these separations were done using the standard proteins. And as you see, we have extra--I mean, we have plotted five consecutive runs on top of each other. And we checked one column first whether it's giving the in--I mean, reproducibility over five consecutive rounds. It is super impossible and absolutely good reproducibility you are getting from this particular column

Now, if we go to the next slide, okay, before that, we tried nine different lots. And we could get exactly very, very reproducible results, which shows that, under these chromatographic conditions, this column has no problem

So, ideally, if we can take care of other things which can spoil the column, we are--we should expect that column life should be pretty good

Next slide, please. So, this is the overlap of the injection number one and injection number 1,000 of one column. Tomorrow, I have a talk on this column lifetime, and that will be the much more elaborated discussion about the column lifetime

But, as you see here, starting from injection number one to injection number 1,000, percentage RSD we calculated for the whole run inequal to 100 because we collected each and every single data. But, when I calculated the RSD, I used every 10th data. So, this is very, very low, very low. I'll give you the results tomorrow in the talk much more in detail

But, even after 1,000 injections, the theoretical plate off PABA was greater than 33,000. It was very, very stable. And as you remember, the total protein content of this particular single run is 0.6 milligram. So, 0.6 milligram I am loading for more than 1,000 injections and still nothing changed in terms of the efficiency

And as you see, the retention time and other asymmetric factors, they are all remaining perfectly same for 1,000 injections

Next slide, please. This is G3000SWXL again because I have shown the standard. Now, it's just a protein. And BSA's a very common protein. I have actually got many questions from different end users. Can you separate the BSA monomer from the dimer and trimer because they use it for different purpose as a standard to begin with

So, what I found, I took another column, which is already run for 1,255 injections using protein standards and other samples. And then I--excuse me--run this particular sample BSA. And as you see, the monomer is very well separated from the dimer and followed by the trimer

Next slide, please. Now, I compared that particular column, which is already done for 1,255 injections with a brand new column from competitor S of exactly the same ID, same length, same pore size. And if you look at the resolution between the monomer peak and the dimer peak, the TSKgel G3000SWXL column is giving much higher resolution compared to the column, which is the competitor column

Next slide, please. So, another question was, how consistent in terms of the metrics we are providing inside the column over the years? So many years back, in 2005, we did similar experiences. As you read, there are a lot of black and white here

But, as you read, in that particular study also, no guard column was used. Buffers and mobile phase was not filtered, just to give extra strays. Protein samples also not filtered. The column was not cleaned. And in that particular study, mobile phase was recycled to give again some more pressure on the column

And as you see, the column lifetime test was excellent. And retention time, asymmetric factor, and the efficiency over 3,000 injections was very good. After that, it started losing because, if we make a limit of 5 percent as the--you know, if we cross 5 percent, we say, "Okay. Now, the column is crossing the limit.

So, after that, it crossed 5 percent. And we could run up to 4,800, 4,800 injections. But, definitely, at that point, column was very low in terms of the efficiency

But, what it shows that the basic metrics, nothing changed over here, nothing wrong, everything is fine. Only thing, depending on the chromatographic conditions and how much care we take, in terms of the factors we have discussed earlier in the slides, we can actually use the column for a long time. There are so many factors

Next, please. Now, this is the profile where we have shown the maximum pressure limit for this column, which is 70 bar. And this is the pressure for the column at the injection number one. And this is the pressure, injection number 1,000, in this particular study

So, over 1,000 injections, the pressure did not change much, though, we gave extra stress because we did not filter or anything. We did not clean

Now, we compared this data with the study which was done in 2005. And this is the data, the pressure after 2,100 injection. Definitely, it is 56 bar, which is more. But, again, it is nowhere close to 70 bar

One thing I will tell you, I have visited many scientists and end users. We always request that many times the system pressure is a factor also. We have to be very careful about the system pressure. We have to optimize the HPLC instrument very well by reducing the dead volume, wide volume, and all of these factors so that you can actually increase the efficiency much more, even if we have, you know, good column

Yes, so, the columns never reached and exceeded the pressure in this lifetime study. It never exceeded the pressure in the lifetime study we did so many years back 2005

Next, please. Now, this is again about the BSA, but it's a different column, just to show that it is extremely reproducible with G2000SWXL column, and G2000SW column is also very much useful for the separation of protein, monomer, dimer, and trimer for consecutive runs

And also, no splitting of the monomer peak was noticed. Sometimes--next slide, please--and sometimes, when you overload the column--next, please--overload the column, you can get a split peak. But, as you see here, we have started with 102 microgram of BSA. The previous slide was 10.2 microgram. So, it is already 10-fold

And then I compared with the 1.2 milligram--1.02 milligram of BSA. And even at that high a load, anyway, it's the analytical column. We are not going to load that much. But, still, as you see, there's no problem in separating the monomer peak with the dimer peak--from the dimer peak. It was giving very consistent result over many repeated injections, using a 10-fold higher load compared to the normal injection load we use in an analytical column

Next, please. Now, about the secondary interaction, which is very important for SEC, there is an article in Bioprocess International which I always request you to read because this is the article where TSKgel G3000SWXL column is very exhaustibly used for this type of secondary interaction issue to study that particular issue

I'm not going into the details. But, this cartoon shows that what happens, a globular protein, which is spherical in nature, is coming into the contact with the surface of the silica surface. When it comes into the contact, if the secondary interaction is very, very high, then it undergoes a conformational change

And that conformational change, as you notice, because of the conformational change, the binding strength between the silica surface with this conformationally changed protein is so high that it does not come out. And you cannot knock it off by any way

So, what we have done, they have used arginine in the buffer, 100 millimolar arginine in the buffer, and found that agninine actually can prevent binding

Now, if you have already loaded the column with the protein and found that the protein totally getting stuck into the column and then you think, "Okay. Let me add arginine into the buffer," it's not going to work because arginine cannot supercede that binding capacity, binding strength

So, what you have to do, you have to have a column, equilibrate the column with the arginine in the buffer for 10-column volumes. And then you load the protein of your interest, and then you will not find any secondary interaction. It will come very nicely out of your column

And I would--just go through that particular article. It's a very nice article they have done using UV as it was I think light scattering also

Next, please. So, it is not just arginine. We have done many different studies using different types of additives. As you see here, we have done the separation using PEGylated proteins. And as you know, secondary interaction is very, very common or expected with the PEGylated proteins

And we have found, over 150 injections, the retention time is gradually shifting to the higher retention time if you don't have any additives. But, if you have additives like 10 percent, just 10 percent ethanol in the mobile phase, again, you are running 150 injections without any retention shift

And we have found--next, please--we have found not just the tunnel [sp]. Even just BSA can also be used to block all the secondary pockets, secondary interaction sites. And then you run your sample

Now, which additive is going to work best for you, that's a trial and error question. You have to try which works best for you depending on the interaction type

Next, please. As you see here--next--as you see here, in this case, effect of the surfactant is also shown from 0.005 percent, here in this case 0.05 percent. And we have used the nonionic surfactant to show that that helps in getting the better peak shape and resolution

Next, please--and also the recovery. So, in general, there are so many different applications we have on this type of interaction. And this is a general guideline, particularly about the mobile phase, how we can enhance the molecular sieving mechanism and reduce the secondary interaction. That is by changing the ionic strength

Low ionic strength promotes ionic interaction. And the higher ionic strength promotes secondary interaction, hydrophobic interaction. So, we should take care of that and play with that and figure it out what is the best chromatographic conditions for the separation

Next, please. But, we have found the type of buffer is also very important. We have a full-fledged separation report applications in our database. If you are interested, just let us know. I'll send you

But, as you see, this is sodium phosphate and potassium phosphate. And this is tris-HCl. In general, we have used these three proteins. And as you see, definitely, depending on the buffer, you are getting the different recovery in terms of separation

Next, please. So, now, I have already told it. But, just to avoid secondary interaction coming from the housing material, I would request instead of using TSKgel G3000SWXL, which is in stainless steel, use the BioAssist, which is BioAssist, means it's assisting in the separation of the bio molecule, which is basically in the PEEK hardware. And you should not expect any problem of that type of interaction

Next, please. So, the conclusion is like this. Silica based, our column is silica based. And it's diol coated to prevent the interaction. It's very, very robust column

No change in the column matrix so far, absolutely no change since the study we did last time in 2005 until now. Lot-to-lot consistency is extremely good. Reliability and dependability of the column is unquestionable

And since the column was studied without the guard column and as I already explained everything in detail, that shows that the column is really, really robust

So, in one line, if I say what is the conclusion? Conclusion is this particular column line is extremely suitable for the separation of the biomolecules in general

Thank you very much. And then I'll take your questions