GlycoWorks RapiFluor-MS N-Glycan Kit by Waters

"Very easy to use; no experience required. Yields high-sensitivity fluorescence and mass spectrometry data in a very short time. Much better than traditional 2-AB labelling. Works also well on simple quadrupole mass detectors (QDa). "

"This kit helps us to get the glycan profile quickly for exiguous sample volume; it enables high quality of results and it is easy to use for chemists, I would like to recommend to those do lots of glycan preparation work "

"If your sample is mAb, it would be easy to use/analyze by following their protocol. But for different materials, it requires modification. It can't be straight use."

"The sample just needs 10 ug. The kit is easy use and high quality, I will recommend to other researchers."

"The GlycoWorks RapiFluor-MS N-Glycan Kit was very easy to use and worked on the first attempt. The data was of a high quality and quantification was very comparable to traditional 2-AB labeling, but in a fraction of the time."

"The following review highlights step alterations we made to increase titre concentrations carrying out the new RapiFluor sample preparation on our low titre Glycan samples. (Previously we used the 2-AB method to do Glycan analysis). Hopefully this will help anyone who has also encountered these issues with their own samples.
We carried out initial Glycan testing using the base method provided with the RapiFluor protocol and found our signals were too low after sample analysis on our Mass Spec. The main issue being that our titre values for samples were below the required specification, because of this we contacted Waters to see what could be done to improve this. The following instructions detail what we did to increase titre concentrations along the process and to therefore achieve increased signal for MS and FLR.
The best way to describe what we did is that we increased Front end loading and low dilution at the back end of the protocol.
Details:
To purify our samples we use GE Protein A spintrap columns. We always spintrap around 2mL of sample for each spintrap column, however even with low titre samples this will still not be enough to increase signal on its own. The final step of the procedure for using the columns is to Elute with 400uL buffer into two 30uL volumes of Neutralizing buffer. Instead we used 300uL of elution buffer into 45uL Neutralizing buffer, this decreasing of dilution should increase the sample titre concentration slightly more before attempting the Rapifluor protocol.
The next alteration is during Step 1 of the protocol (Rapid Deglycosylation), part 2 and 3 of this step details using water with Glycoprotein. However, with concentrations below 0.75mg/mL we used no water and used 22.8uL of the Glycoprotein. After this step follow the process to Step 3 (HILIC clean-up)
At Step 3, part 7, the protocol states to Elute with three 30uL volumes of SPE elution buffer. We did this, however, after the volumes were collected we dried the samples down by Vac centrifuge for 1 hour 30 minutes before starting part 8.
Then when we did part 8 instead of diluting with 100uL DMF and 210 ACN, we reconstituted with 9uL water, 10uL of DMF and 21uL ACN. This giving 40uL of your glycoprotein sample.
That is what we did to increase the sample concentrations throughout the protocol. The final thing we did was to inject 20uL sample volumes for analysis on the MS.
Below I've detailed the difference in signal strengths between our initial run using the protocol word for word, and then for the altered procedure.
Initial run: Sample concentration 0.25g/L TIC signal 4.38e4 FLR signal 10,000 EUx10e4
Altered run: Sample concentration 0.20g/L TIC signal 4.25e6 FLR signal 5,000,000 EUx10e4
The instrument was optimized to deal with this method analysis.
The advantage of this method is that it is very quick to use, even with the Vac drying stage (it still beats the 3-5 day 2AB procedure formally used).
The profiles given were very reliable and reproducible to the reactor pattern given using our previous method.
"

"RapiFluor-MS has provided the ability to characterize N-glycans with greater precision and confidence. It has also enabled the identification of previously unknown glycan structures and modifications, thanks to its compatibility with MS technology. Novel approaches such as RapiFluor are sometimes met with apprehension, due to lack of experience with the technology. However, RapiFluor-MS has demonstrated its utility in a number of application areas with confidence and experience continuing to grow on a weekly basis. Adoption of novel labelling technology can require extensive evaluation and comparison to legacy workflows. Information on how RapiFluor-MS compares to traditional reductive amination (2AA, 2AB, and APTS) as well as alternative separation techniques such as CE-LIF would further support RapiFluor-MS as complementary approach for glycan characterization. "

"This product enables to prepare labeled glycans within one day with a minimum of initial material for a decent signal. The quality and reproducibility of the results are satisfying and align with previous classical digesting/labeling kit. The kit is easy to use and the procedure well described, but require some equipment such as a negative pressure manifold etc. On the other hand, the different tubes provided in the kit are not very suitable for the volumes pipetted. The after sales care and follow up is great and the questions asked are always answered in a short time. Finally, the value for money is decent, but the kit is of course slightly more expensive than previous kit. The gain of time is indisputable for few samples but decrease for a full 96 well plate "

"This product enables to prepare labeled glycans within one day with a minimum of initial material for a decent signal. The quality and reproducibility of the results are satisfying and align with previous classical digesting/labeling kit. The kit is easy to use and the procedure well described, but require some equipment such as a negative pressure manifold etc. On the other hand, the different tubes provided in the kit are not very suitable for the volumes pipetted. The after sales care and follow up is great and the questions asked are always answered in a short time. Finally, the value for money is decent, but the kit is of course slightly more expensive than previous kit. The gain of time is indisputable for few samples but decrease for a full 96 well plate "

"It is very fast, simple, highly sensitive in FLR and more exciting is about its MS signal sensitivity with which we could identify so far unannotated glycans"

"The process is very simple; however, the kits lack of flexibility in terms of number of samples need to be analyzed (need to at least use 8 wells). When a large number of samples need to be run, sample preparation time will increase significantly. The cleaning step using vacuum manifold is tricky sometimes to obtain good recovery. The fluorescent signal is excellent and glycan separation is very good which we really liked. The mass spec signal acquired with QDA detector however is somewhat weak and hopefully can be improved. "

"This kit is amazing. Not only the data I got was highly reproducible but the kit makes everything high throughput. You can run a 24 or 96 samples in less than a day. The average glycans profile can take up to 72 hours in sample prep time. GlycoWorks RapiFluor gets the job done in 2hours. It is also MS friendly. You simply can't get any better than that. "

"Simple to use great support, Excellent value for money."

"it is easy to use with great MS signal for the glycan structure identification."

"Kit is highly effective, easy to use, saves lots of time and troubles as compared to previous available options of glycan analysis. "

"This product is fundamentally changing glycan analysis. It provides fast and effective sample preparation, convenient labeling and clean-up and reliable results. It made glycan analysis finally user-friendly. It also provides enhanced fluorescence and mass spectrometry sensitivity the same time. "

"I start with far less protein than I use to start with and I get comparable results. The protocol is easy to follow and quick to run. Waters respond promptly to issues and they are effective at resolving them. It represents value for money because if you do not need to run 96 samples, you can buy a kit to run 24 samples. Super!"

"Much more sensitive with fluorescence and also more sensitivity with MS. Previous 2-AB protocol took ~7 hours this can be done in ~2,5 hours"

"Easy to use, for quick analysis of N-glycans. Labelled glycans can be detected by Mass Spectrometry operating in positive ion mode. Reproducible results."

"The RapiFlour-MS kit from Waters has improved the workflow on how we assess the glycosylation pattern of our protein based products. The kit is fast and robust allowing for MS quantifiable results, hence strengthen our confidence in our analytical results. This kit has given us a confidence that we move the kit into a validated environment with the intended purpose to use the results as a QC release test. Further, the kit allows for analyzing smaller lots of samples, compared to older kit that forces you to analyze 96 samples or waste the rest of the kit, thus decreasing the price of the individual analysis. "

"The rapiFluor kit is an ideal method for easy and quick measurement of glycoproteins. Because of the speed of the method, this kit is ideal for labs that have a large amount of samples."

"We have tried the GlycoWorks RapiFluor-MS N-glycan kit for some of our products and I was really pleased with the result. Although it was the first time we tried the kit, it really turned out to be very quick and easy to use. What especially was amazing was the similar peak profiles and sensitivity we obtained when comparing fluorescence detection and MS. This made us to decide that in the future we do not need fluorescence and only use MS for both relative quantification and identification."

"N-Glycan analysis has become a breeze with this new RapiFluor kit! Quite a bit of optimization was needed for our specific needs, but upon completion, the kit has worked wonders for us. We have used two lots of kits and they both have worked well. I like the option to buy a smaller kit to reduce our waste."

"The GlycoWorks RapiFluor-MS N-Glycan Kit streamlines the workflow. It reduces complicated, time consuming sample preparation and we could have the result ready in a day including uPLC separation."

"I have only run a number of standards using this instrument because this is the first time that my lab has attempted to quantify and study glycosylation using LC-MS. As someone who hasn't worked with HPLC or LC-MS before I was impressed with the after purchase support offer from Waters and the relatively simple product preparation. I will be interested to see the sensitivity that can be obtained with this instrument even in our complex lysate samples."

"I was very impressed by the ease of using this product. It was very easy to set up and really reduced the time of our previous method by an entire day. This product will greatly improve our workflow. In addition, Waters representatives were swift in scheduling a demo and helping us every step of the way. Very happy with this purchase."

"This kit is easy to use and provides highly reproducible results, both with fluorescence detection and MS analysis. Moreover, sample preparation can be performed in short amount of time. "

"the product is easy to use and it has shortened sample preparation time significantly. However, each reaction is still much more expensive as compared to 2-AB labelling glycans, hence it is mainly reserved to analyze more complex samples only. "

"I have recently begun evaluating the RapiFluor kit. I've found the instructions and the kit contents very easy to use, and haven't had to amend the kit instructions for our own use. After an initial evaluation the glycans were well separated, further testing will confirm whether reproducible results are achieved. Overall I am happy with this kit. "

"Easy to use, fast sample preparation, it really takes only 30-40 min. Nice FL and MS signals even for the minor glycans."

"The positives compared to other available... 1. Ease of use 2. Number of samples which can be analyzed in one go 3. Time taken to complete the analysis"

"Rapifluor kit is very much useful for characterizing the glycan profile for Novel and Biosimilar antibodies in high throughput analysis. This kit sensitivity is excellent if we are using LC/MS. We are finding difficulties in eluting the sample without a positive pressure manifold, instead if the kit also designed(plate) to use centrifuge (plate holding rotor) for washing and elution will be great added advantage. The repeatability is excellent, we found little less fluorescence signal while using only LC with fluorescence detector using this kit when compare to regular 2AB labeled glycan analysis."

"Kit is used for glycoprofiling of recombinant proteins and mAbs. Applications are to support, clone selection, cell culture process development and characterization and release of drug substance. Review: We find the kit easy to use and highly efficient. We are experiencing problems with variability of retention times within runs when using our H-class bio system with RapiFluor-MS standard method. This in combination with insufficient peak capacity and resolving power in regions of (Man5, G1, G1F-GN) and (G2, Man6, and more) are slows down data evaluation and introduces ambiguity in peak assignments. Some of these ambiguities are inconvenient and may lead to us drawing the wrong conclusions for the experiment at hand. Apart from these comments we are very satisfied with the performance of the kit and with ease of use and through-put. "

"We used the kit to analyze glycans from mAbs. What was particularly interesting for us was the claimed higher LC-MS sensitivity. Overall the kit was relatively easy to perform and delivered what is promised. "

"The Waters kit has been wonderful out of the box. It is very easy to use and scale, especially when compared to other brands that we have used in the past and have tested out recently. The time from prep to sample completion is extremely quick and well thought out, and the data is comparable with other tests and comes out crisp and clear."

"Overall the kit is exceptional. The instructions are easy to follow and everything is labeled perfectly. The requirement of additional equipment (outside of the UPLC) is minimal and should be common in most analytical laboratories set up for protein characterization, greatly reducing any additional costs. The kit is easy enough to use that you feel you aren't really doing enough for a typical digestion and analysis, however the results you get speak otherwise. The profiles are well resolved and reproducible. The sensitivity is so great that you can quantify differences in glycan species and varying amounts therein whether they be lot to lot or species to species comparisons. The kit can be purchased for small or large throughput allowing for flexibility of sample population size for development work, validation, or QC testing of a final product culminating to little or no waste of leftover reagents. Clearly a new benchmark in protein characterization has been set. "

"The kit is amazing, very easy to use and follow the instructions. The results really reproducible, I have done this assay few times using same sample and every time I got similar results."