GE Healthcare on Michael’s Domain

Showdown: GE Healthcare's Äkta versus Bio-Rad's NGC

Tue, 07 Feb 2023 00:00:00 +0000

I have been purifying proteins since 1996. I worked on an Äkta Explorer until 2015, when we upgraded to the Äkta Avant . In 2020, my lab moved, and we inherited a Bio-Rad NGC , which we have been using now for 2 years.We encountered many problems with the Bio-Rad NGC. At first, I thought that this might be normal when switching systems. I expected the problems to disappear one by one. After all, we also had problems when we switched from the Explorer to the Avant.However, even after two years and dozens of purification runs, the problems with the NGC don’t seem to stop. Whenever ẃe solve a problem, a new, previously unknown problem appears. And differently to GE Healthcare, Bio-Rad’s customer service is not even close to what we have experienced with GE Healthcare. When our IT could not connect the Äkta to our university’s network, GE Healthcare sent an engineer from their Munich crew to Helsinki to fix the problem. Appreciating the learning curve, GE Healthcare also offered free participation in one of their courses for somebody from our team. And their support was not limited to the warranty period! They really wanted us to be happy with their device. We don’t experience the same amount of support from Bio-Rad. It always feels like we have to coerce them into solving the problems we have with the NGC, and their response time is well below any customer expectations.This is the first of several blog posts about Äkta versus NGC. I hope will find the time to write in more detail about all our issues over the next few months.We decided in 2015 that we finally needed a new FPLC. Our Äkta Explorer was reaching end-of-life, and we had received about 100k funding to renew the FPLC of our core facility . The device was expensive enough that we needed to go through the official tendering process (at the time, the limit was 30,000 €, but it has been increased to 60,000 € by now). There were only two contenders: The Bio-Rad Discover NGC and the GE Healthcare Äkta Avant. The Bio-Rad was cheaper (82,810 € versus 95,470 €), but we decided to purchase the GE Healthcare device. One important reason was that all our users had been using the Äkta Explorer and its Unicorn software. Switching would simply be disruptive and require lots of support and time from our side. However, there were also technical reasons that made us prefer the Äkta over the NGC:Äkta Avant’s advantages

Increased resilience

Tue, 27 Nov 2018 00:00:00 +0000

I strongly believe that companies are genuinely interested in getting critical feedback. At the very least, it should be part of their corporate survival instinct. When dissatisfied customers simply switch to an alternative vendor without giving feedback, the damage is already done. That’s why I always give feedback if a product does not meet my high-quality expectations.However, this time I am writing about a product I have been very satisfied with, namely the new GE Healthcare Superdex 75 Increase 10/300 GL . It is a gel filtration column and an iteration of the previous Superdex 75 10/300 GL , which we have been using for the last 30 years. The biggest advantage of the “Increase” is the higher pressure-resistance. A few weeks back an overworked grad student forgot to close the column after use and returned it to the fridge, where the 20% ethanol slowly evaporated over the following 2 weeks. The fridge is ventilated and when I needed the column, the damage was already done: There was a perhaps 8 mm gap and a dry zone of approximately once inch had been developing. I immediately filled up the dead volumes with degassed 20% ethanol and started a very slow run (0.05 ml/min) for several hours, after which is switched to degassed water and then to buffer. After letting it run for about 2 days the gap was reduced to about 3 millimeters. The remaining gap was removed by adjusting with the top adapter (I needed to screw it down as much as possible).Now I needed to test the “repaired” column. I did both a functional test (separating two proteins in PBS) and the acetone test (injecting 100 µl 2% acetone in water). I was massively surprised when the aceton test showed about 19000 theoretical plates (which is more than we ever got with our old Superdex 75 columns). And the separation of the two proteins (RNaseA and BSA) showed that the column is still fully functional. However, we cannot tolerate any gel compression, since the ability of the adapter to correct for it is maxed out (GE Healthcare used to produce a longer adapter, which would allow us to compensate even further, but they chose to discontinue this product).Both the grad student and myself were relieved after getting these results because buying a new column would have set us back by 2200€ and since we have no dedicated funding to operate our core facility , I would have been forced to offload this expense to the grad student’s laboratory. Unfortunately, I did not take a picture of the damaged column as my instinctive reaction was to immediately start the rescue. However, the column in the picture below is the damaged column after the rescue operation was complete.

Purification and Characterization of Recombinant Proteins

Mon, 20 Mar 2017 00:00:00 +0000

We are organizing (again) a practical hands-on protein purification course from December 4th to 20th. We maximally can accommodate 16 participants, which will form groups of 2 to 4 participants. Each group needs 3 full days to go thru the practical exercises, but day 3 of the course will be overlapping with day 1 of the next group. Venue is Biomedicum Helsinki, rooms A516a1 (where the machinery is) and B318a/b (our lab). We will purify a protein (VEGF receptor 3) using a two-step protocol (affinity chromatography + gel filtration) on the the Äkta Avant FPLC device. On the third course day we’ll assay its interaction with its ligand (VEGF-C) on the ITC (isothermal calorimetry) device. We have given a similar course in 2015 ( http://www.helisci.fi/hbgs/FPLC2015/) . This new course ( https://courses.helsinki.fi/en/DPBM-135/120171139 ) will have a similar structure, but we expand it by one day to analyze protein interactions making use of the new isothermal calorimetry device of our B3P core facility. The documentation and results will be also available from here . Since we can run maximally two samples at a time (we have “only” two FPLC machines), we will have to split the participants into groups (of 2-4 students/group) and repeat the 3-day course several times depending on the number of participants.

Unicorn 7-Benutzer-Einrichtung erfordert manuelle Intervention in einer Netzwerkbenutzer-Umgebung

Fri, 17 Jun 2016 00:00:00 +0000

Unicorn, Äkta Avant, Äkta Explorer, GE Healthcare, Windows, Netzwerk-Authentifizierung, Benutzer-Einrichtung, Methode, Resultat, Wissenschaft, ProteinaufreinigungSince we are operating our Äkta Avant in a multiuser environment, we need to separate the methods and results of the different users. By default, every network user is at the moment able to see every method and every result that has been generated on the machine by any other network user. This is a considerable privancy and security issue as a malicious user could delete (or even worse: modify) methods and results.Our Äkta is set up in a way that allows users to log into the Unicorn 7 computer with their university login/password via the regular Windows network authentication mechanism. However, if several people share the responsibility of a run, this setup becomes impossible as they would need to devulge their passwords to each other. Hence we have created a local account which can be used by users who wish to share the operation of the Äkta.After logging into Winodws, users still have to log into the Unicorn 7 software, which users do with their university login and password (“Windows authentication”). Using this setup, every user is able to see all methods and results, which is not acceptable.When setting up a new user with Unicorn version Access>Folders") and exactly which folders were accessible by that user and the user would see only his/her own methods and results. Since the folder structure under Unicorn 5 was a folder structure of the Windows file system, users could always copy methods and results from one folder to another and thereby make them available despite the limitations set by the Unicorn 5 program.When I first read that Unicorn 7 supports Windows network authentication, I had hoped that we would be able to avoid the painful user setup which we had to go thru for each user on the Äkta Explorer. However, the pain continues as setting up the user privileges once for a group doesn’t give us user isolation.Firstly, one cannot restrict access of individual users in Unicorn 7, but only access of groups. We had to create one Access Group for each network user, add the network user to this group, create a separate home folder for the group and then restrict the folder access to this home folder. Hundreds of clicks were required for a handful of users since the default is no access to anything and every single privilege check box needs to be enabled except for the admin privileges.In that respect, Windows (and every other OS) is way smarter than Unicorn. If a university employee logs into a machine that he or she has never been logging into before, it creates all the necessary default local folder structure automatically and mounts that users private home folder without granting access to everything other employees have been doing on that specific machine. I think that should be an option on Unicorn as well. Maybe it is and I just can’t figure it out?Another big drawback of the above described method of separating each user into an own access group is that login into the Unicorn program becomes a major ordeal: In addition to writing user name and password the user has to select the correct access group for the login to be successful. And even worse: In our setup we cannot avoid that every university employee belongs to two access groups: A manually created access group for each user for user separation and the “default” which works via the Windows network authentication - maybe Kerberos?). Hence, if users do choose the default access group (which is called “Users” in our case), they are able to log in, but they don’t see their methods and results.There are two reasons we cannot delete the “Users” access group: One is the mandate of the faculty and secondly (and we have tried), we cannot delete it anymore as many people have already created methods and generated results being in the access group “Users”. Thus UNICORN prevents us from deleting this account. I am working on this problem: I should be able to access directly the underlying MS-SQL database in order to change the ownership of the methods and results. However, GE was not exactly forthcoming when I was asking about access right handling. The answer was:The DB access credentials in a standalone UNICORN solution are encrypted and are not public. If you had an enterprise solution (hosting your own (SQL server) DB) you would have control of the credentials and in theory you could extract the wanted information (the format is something that you have to figure out by yourself and is not supported by us). You can upgrade your solution to an enterprise if you want.This sounds worse than it is, because we have physical access to the MS-SQL server and pulling out the access credentials seems not very difficult. But it takes my time to find the exploit to “break into our own system” and that is what annoys me. However, according to Lisa Bromark from GE, the 7.0.2 update seems to correct this issue:UNICORN can be configured to use a new database password. It is possible to generate an encrypted password or to enter an already encrypted password. This is done by running the UNICORN Service Tool after UNICORN installation.However, it is unbelievably difficult to get the update (at least it seems to take weeks). Distribution is apparently still via optical media and snail mail. I think the last time I got myself software via a CD/DVD was more than 10 years ago. However, GE told me that they are just moving UNICORN software updates to “electronic distribution”. Welcome to the 21 century!

Unicorn 7 user separation requires manual intervention in a network user environment

Mon, 09 May 2016 00:00:00 +0000

Since we are operating our Äkta Avant in a multiuser environment, we need to separate the methods and results of the different users. By default, every network user is at the moment able to see every method and every result that has been generated on the machine by any other network user. This is a considerable privancy and security issue as a malicious user could delete (or even worse: modify) methods and results.Our Äkta is set up in a way that allows users to log into the Unicorn 7 computer with their university login/password via the regular Windows network authentication mechanism. However, if several people share the responsibility of a run, this setup becomes impossible as they would need to devulge their passwords to each other. Hence we have created a local account which can be used by users who wish to share the operation of the Äkta.After logging into Winodws, users still have to log into the Unicorn 7 software, which users do with their university login and password (“Windows authentication”). Using this setup, every user is able to see all methods and results, which is not acceptable.When setting up a new user with Unicorn version Access>Folders") and exactly which folders were accessible by that user and the user would see only his/her own methods and results. Since the folder structure under Unicorn 5 was a folder structure of the Windows file system, users could always copy methods and results from one folder to another and thereby make them available despite the limitations set by the Unicorn 5 program.When I first read that Unicorn 7 supports Windows network authentication, I had hoped that we would be able to avoid the painful user setup which we had to go thru for each user on the Äkta Explorer. However, the pain continues as setting up the user privileges once for a group doesn’t give us user isolation.Firstly, one cannot restrict access of individual users in Unicorn 7, but only access of groups. We had to create one Access Group for each network user, add the network user to this group, create a separate home folder for the group and then restrict the folder access to this home folder. Hundreds of clicks were required for a handful of users since the default is no access to anything and every single privilege check box needs to be enabled except for the admin privileges.In that respect, Windows (and every other OS) is way smarter than Unicorn. If a university employee logs into a machine that he or she has never been logging into before, it creates all the necessary default local folder structure automatically and mounts that users private home folder without granting access to everything other employees have been doing on that specific machine. I think that should be an option on Unicorn as well. Maybe it is and I just can’t figure it out?Another big drawback of the above described method of separating each user into an own access group is that login into the Unicorn program becomes a major ordeal: In addition to writing user name and password the user has to select the correct access group for the login to be successful. And even worse: In our setup we cannot avoid that every university employee belongs to two access groups: A manually created access group for each user for user separation and the “default” which works via the Windows network authentication - maybe Kerberos?). Hence, if users do choose the default access group (which is called “Users” in our case), they are able to log in, but they don’t see their methods and results.There are two reasons we cannot delete the “Users” access group: One is the mandate of the faculty and secondly (and we have tried), we cannot delete it anymore as many people have already created methods and generated results being in the access group “Users”. Thus UNICORN prevents us from deleting this account. I am working on this problem: I should be able to access directly the underlying MS-SQL database in order to change the ownership of the methods and results. However, GE was not exactly forthcoming when I was asking about access right handling. The answer was:The DB access credentials in a standalone UNICORN solution are encrypted and are not public. If you had an enterprise solution (hosting your own (SQL server) DB) you would have control of the credentials and in theory you could extract the wanted information (the format is something that you have to figure out by yourself and is not supported by us). You can upgrade your solution to an enterprise if you want.This sounds worse than it is, because we have physical access to the MS-SQL server and pulling out the access credentials seems not very difficult. But it takes my time to find the exploit to “break into our own system” and that is what annoys me. However, according to Lisa Bromark from GE, the 7.0.2 update seems to correct this issue:UNICORN can be configured to use a new database password. It is possible to generate an encrypted password or to enter an already encrypted password. This is done by running the UNICORN Service Tool after UNICORN installation.However, it is unbelievably difficult to get the update (at least it seems to take weeks). Distribution is apparently still via optical media and snail mail. I think the last time I got myself software via a CD/DVD was more than 10 years ago. However, GE told me that they are just moving UNICORN software updates to “electronic distribution”. Welcome to the 21 century!

The logic of the Äkta Avant fraction collector

Mon, 02 May 2016 00:00:00 +0000

After we solved all the [teething troubles with our Äkta Avant]((/en/akta_avant), we finally dare to let customers use it. Our customers are very heterogeneous covering complete novices to FPLC and experienced Äkta Explorer users (and everything in between). When we have been giving feedback to GE, our perspective is obviously biased towards a certain type of user. However, taking care of customers is giving us now a new perspective since we get confronted with the usability problems that they cannot solve by themselves. Here I just want to mention one stumbling stone, that has repeatedly brought up to us: the rationale behind the operating mode of the fraction collector. Unlike in the older systems, the fractions collector cannot be manually reset to “First position” or to any arbitrarily defined position as was possible e.g. under Unicorn 5.It took us ourselves quite a while to get used to the internal logic of the fraction collection process and we needed guidance from GE. The fact that the system is not behaving intuitively is underlined by the fact that some of the answers that we received from GE experts were incomplete (leading for us to some unpleasant sample losses). Finally we received from GE support a table that describes the behaviour of the fraction collector (see below). However, even that table is incomplete and we have added a few lines that describe some non-standard situations for which the table does not provide an answer. These changes and additions to GE’s description have been marked in red.

HiLoad 26/60 Superdex pg gel filtration chromatography column performance

Mon, 11 Apr 2016 00:00:00 +0000

The most common protein purification technique that we use is gel filtration (also called size exclusion chromatography). In gel filtration, proteins are separated by their size (or more correctly by their “Stokes radius”, which is largely determined by their size and shape). For gel filtration of large protein amounts, we bought GE Healthcare’s HiLoad 26/60 Superdex 200 prep grade about 9 years ago (the 26/60 has been replaced by HiLoad 26/600, but both are almost identical). With this column you can very cleanly separate two proteins that have a size difference of 100%. Therefore it is suitable to separate monomeric from dimeric versions of the same protein. The columns “expiry date” was 2012-03. Our experience is that under proper handling, such a column can easily reach a life span of 10 to 15 years (we don’t use it very often, we store it in 20% ethanol, if we don’t need it for longer periods of time, we keep it at +4°C).

Review of GE Healthcare's Äkta Avant 25

Wed, 02 Mar 2016 00:00:00 +0000

About one year ago, we secured funding in an internal faculty call to replace the old Pharmacia Äkta Explorer 100 FPLC machine of the Protein Production and Purification core facility . The Äkta Explorer had been purchased in 1996(?) when the Swedish brand Pharmacia still existed. In 2014, the worse case scenerio happened and both monochromator and Xenon flash lamp broke almost simultaneously, which left us with a bill of almost 10000€. Hence we wanted to replace the machine with a contemporary model. There are only two companies offering serious devices in this space, which is GE Healthcare ( Äkta product line ) and BioRad ( NGC product line ). GE Healthcare inherited the Äkta brand from Pharmacia via multiple mergers, while BioRad is a relatively new contender with their NGC models, which they released only a few years back.We opted for the Äkta Avant 25 . We definitely wanted to have a cased, refrigerated fraction collector. And we wanted to have a familiar user experience. We often run automated purifications and we do not like our proteins to be for longer times at room temperature in open tubes into which bacteria and dust from the air can enter.By now, we have operated the Äkta Avant for about half a year and we have meanwhile a good idea how it compares to the Äkta Explorer. Even though the Avant is more modern than the Explorer, all users of our core facility still use the Explorer. They are familiar with the user interface of Unicorn 5.11 and seemingly have no interest in learning the - admittedly - more complicated UI of the Avant. But the other reason it was only in internal use so far was the fair amount of problems that we have encountered. With our old Äkta Explorer, we have never seen so many problems in such rapid succession. I don’t know whether our situation is typical (according to GE Healthcare’s representatives it is not). Nevertheless, here are the issues that we encountered:

FPLC Protein purification course

Mon, 04 Jan 2016 00:00:00 +0000

Eight postgraduate students registered for the FPLC protein purification course , which took place in December. If I learned anything, than that protein purification during a course should be done ALWAYS with a protein and a protocol, that has been used before successfully MANY times. Student-provided proteins are a great source for learning, but the time restraints of a course format did not allow us to finish the purification of these proteins during the course.Technical Problems with the new Äkta Avant 25In addition, the Äkta Avant 25 , that we newly purchased from GE Healthcare this summer, broke TWICE during the course. First the controlling computer broke (RAID failure). HP delivered the replacement drive within 24 hours and after the RAID had rebuilt itself, we could continue the course. However, during the first run after this incident, the Äkta ran into an overpressure problem. We identified a faulty flow restrictor as the cause, but we did not want to continue as we have had severe problems with air bubbles in previous runs. Therefore we performed the runs on the old Äkta Explorer 100 . GE Healthcare quickly had their service engineer check out the system, but since he did not have a spare with him, we had to wait until Monday 14.12. until the Äkta Avant was again fully functional.What we purified: soluble VEGFR-3 (VEGFR-3/Fc) and HepsinWe did purify soluble human VEGF receptor-3 (the first three domains of its extracellular domain connected to the constant Fc part of human IgG). This is a purification that we have done many times. It is equivalent to the purification of antibodies using Protein A sepharose.We had prepared in advance conditioned cell culture medium. We produce most of our proteins in insect cells (mostly Drosophila S2) and the VEGFR-3/Fc had been secreted by the S2 cells into the medium after induction of the metallothionein promoter with 1 mM Cu2+ for about 4.5 days. The preparation of the medium for purification consists only of 1) getting rid of the cells by centrifugation and 2) filtration to remove precipitates and other small particles that might clog the column. There is no need to adjust the pH.Rapid neutralization after low pH elution IS IMPORTANTWe ran the medium over a disposable 5-ml HiTrap recombinant Protein A column over night at about 1ml/min and eluted with a low pH buffer. The eluted 2-ml fractions were immediately neutralized with 400µl 1M Tris pH 8.5. Here we made a small mistake during one of the two purifications. GE Healthcare had not released the casettes for the 5-ml-collection tubes (they still have not done so even though they did promise them already for September 2015) and we used 15-ml Falcon tubes to collect 2-ml fractions, into which we had pre-aliqotted 400 µl of the neutralization solution. However, the mixing in these tubes was not efficient and the prolonged exposure to low pH resulted in a partial damage to our protein. This can be seen when comparing the size exclusion chromatograms of Group 2 versus Group 4: For Group 4 the first peak (aggregated protein) is much larger and more heterogenous compared to the same peak for Group 2.In fact, when VEGFR-3/Fc is eluted by low pH from protein A columns, it always precipitates at higher concentrations soon after elution, but dissolves again upon neutralization. This did not happen in the fractions 5.A.3 and 5.A.4 (Group 4) due to the inefficient mixing of elutate and neutralization buffer in the 15-ml-Falcon tube (the fraction size of 2 ml was probably to blame as well; 1 ml would have been better). During the run for Group 2, we removed the tubes immediately after the run had ended and thereby mixed the buffers, while for Group 4 the run finished during night time and the eluate remained largely unmixed until the morning.Alternatively, we could have eluted with a highly concentrated chaotropic salt at near-neutral pH (which we’ll do next time in case we have an automated run where the elution happens in the middle of the night). Pierce offers a (proprietary) “gentle” elution buffer of pH 6.6. I don’t know the Pierce buffer composiiton, but it is a highly concentrated solution of some chaotropic salt. 3M potassium/sodium thiocyanate or 4M magnesium chloride in buffered solutions around pH 7 are frequently used chaotropic salts for this purpose.HepsinThe student-provided proteins were challenging. First, their concentrations in the starting material was very low. While we can see clearly the protein when the VEGFR-3/Fc conditioned medium is run on a PAGE gel and stained with Coomassie, no such band is visible for the Hepsin. In addition, it appeared that a significant fraction of the protein seems not to contain the histag (anymore) and therefore is not captured with the first purification step. The fraction of Hepsin-H6 that does bind to the Ni2+ sepharose elutes already at an imidazole concentration of 20 mM, which makes washing the column challenging. The Hepsin with the longer histag (H10) survives the 20 mM imidazole wash, but it suffers also from low expression levels.Below are the chromatograms of the individual runs and the annoteded images of the Comassie-stained PAGE gels. The detailed protocol for the operation of the Äkta Avant 25 is still under preparation…

How to convert EMF files into SVG format

Tue, 06 Mar 2007 00:00:00 +0000

The need for this appeared because the Unicorn software that controls our Äkta Explorer can only export the raw chromatographic curves in EMF format. Pathetic. After trying out various things, I went with OpenOffice. OpenOffice Draw can import EMF files and save as SVG. Other applications capable of opening EMF are FreeHand and CorelDraw (tested on Macintosh). However, FreeHand cannot export as SVG.