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Intermittent Low recoveries for MAXPAR Conjugation

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claudechew

Participant

Posts: 10

Joined: Tue Nov 04, 2014 5:13 pm

Post Fri Jun 01, 2018 8:41 pm

Intermittent Low recoveries for MAXPAR Conjugation

Dear CyTOF Forum users,

I have been successfully conjugating antibodies now for about 5 years with anywhere from 60-80% recovery and all of a sudden about 6 months ago I started experiencing intermittent low recoveries for antibodies within certain batches of conjugations. I typically conjugate around 4 antibodies at a time and 3 out of 4 will have good recovery and then one will have very low recovery, around 10% or less. This varied between suppliers of antibodies, and there seemed to be no pattern. I started checking the antibodies pre-conjugation with nanodrop to make sure that we were being sent the right amount of antibody from the companies and most of the time this has been true.

So in an effort to resolve this, I threw out all my old conjugation buffers, replaced the TCEP, switched to 30k spin columns to try to ensure better recovery, made sure my water bath was actually at 37 degrees C, and I performed a batch of conjugations today with the same, intermittent, random loss. One antibody recovered at around 60%, one at around 30% and one essentially nothing.

Wondering if anyone has any suggestions to troubleshoot what is going on????

Thanks,
Claude
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ErinSimonds

Master

Posts: 50

Joined: Tue May 13, 2014 8:04 pm

Post Fri Jun 01, 2018 9:32 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

We've seen something like this as well. At one point got paranoid enough to ask our best technician label 8 batches of 100 ug of a cheap antibody in parallel. One of the 8 reactions had ~0% yield, while the other 7 looked great.

We haven't nailed it down beyond a shadow of a doubt, but we suspect the culprit is damaged filters. Specifically, the Amicon Ultra (V-shaped) filters have a fragile white membrane film on the inside. If this gets scraped with a pipet tip, it can create an imperceptible hole that allows the liquid to bypass the filter membrane.

We've started doing a few things:

1) We spec every vial of antibody from the manufacturer on the Nanodrop to verify that they delivered what is claimed on the label. It's surprising how often this is wrong. If you actually put 50 ug into a what you thought was a 100 ug reaction, it will obviously kill your yield. You're probably already doing this, as it has been the recommendation for a long time, but I want to re-emphasize it.

2) We now check the integrity of each filter column with the following protocol: Add 400 uL MilliQ water, and spin at 12000g for 30 seconds. There should be ~150 uL remaining. If the level is lower than this, we discard the filter. Spin 5 more minutes to dry the filter.

3) Our in-house protocol now has a warning in bright red text about never touching the pipet tip to the white membrane inside the spin filter. Being mindful of this has helped.

After implementing these reforms, we have labeled >17g of antibody (170 polymer reactions) with one failed reaction (14% yield on a 300 ug labeling reaction), and for that one, the stock antibody had a very weird absorbance spectrum.

This may not be the gremlin in your lab, but wanted to pass along our experience.

ES
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claudechew

Participant

Posts: 10

Joined: Tue Nov 04, 2014 5:13 pm

Post Mon Jun 04, 2018 1:13 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi Erin,

Thanks so much for sharing your experience with me, this must be a relatively recent thing with the possible issue with membrane integrity, I haven't changed my pipetting technique but I will typically try and wash the white membrane down particularly during the combining of the partially reduced antibody with the metal polymer complex and then followed by the 1.5 hr incubation.

I'll definitely try implementing these and see if that helps. I wonder too if it is just an issue with the Amicon filters? I have been thinking of switching over to the Pall 30K spin filters since they are also a little cheaper and they have that flat membrane, although it does look like it's also coated with a white membrane. But based on my experience with the 3K spin filters that I use for the metal+polymer spins it seems like they may be a little more durable.

Thanks again, I will see how the next batch goes with these things in mind!

Thanks,
Claude
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Jun 04, 2018 4:26 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi Claude,

I personally haven't had recovery issues lately, but I did help a lab in Colorado troubleshoot their recent issues.

They also tracked it down to spin filters. Hopefully one of them will respond to this message with more details, but I did want to add my voice to the possibility of spin filters being a problem.


I also had one bad lot of spin filters about 3 years ago; I believe out of 15 conjugations, 7-8 of them had recovery under 30%. I corresponded with Millipore; they were skeptical, but I was able to return the remaining filters out of the box (for their investigation) and get a replacement box at no additional charge. So, you might check in with them....



Mike
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fcl54643

Contributor

Posts: 21

Joined: Mon Nov 02, 2015 2:13 pm

Location: Greater Philadelphia Area

Post Mon Jun 04, 2018 5:15 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi Claude,

We too have had recovery issues in the past 6 months (it started in Nov-Dec). We tried a new batch of 50k Millipore filters with no success, and moved on to troubleshooting our antibody partial reduction step (new batch of TCEP, different incubation times, different concentrations) with minimal or no improvement. We eventually switched to 30k Millipore filters, and that appeared to have fixed our problem. We now also prime the 30kDa filters with R Buffer before using them. Our antibody recovery is back to old levels: 60-80%

Kind Regards,
Florence
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claudechew

Participant

Posts: 10

Joined: Tue Nov 04, 2014 5:13 pm

Post Mon Jun 04, 2018 6:12 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi Mike,

Yes I actually have asked for 2 lots of replacements, one for a 50K batch to no avail and also for a 30k batch, both didn't help and also Millipore claimed no errors or issues reported so far, although I guess what I could have said may have been the first issue reported...

Hi Florence,

I might try priming with R-Buffer, do you add say 200-400ul and then quick spin them to prime them?

Thanks,
Claude
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ErinSimonds

Master

Posts: 50

Joined: Tue May 13, 2014 8:04 pm

Post Mon Jun 04, 2018 6:44 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

I also bought a new batch of 50kDa filters hoping to remedy the issue. It was a different lot #, but we still had problems with the new lot. I didn't try the 30k filters.

A word of caution on the flat-bottom Pall NanoSep filters: DVS Sciences recommended these filters in the very early days of MaxPar/CyTOF, before the commercial release of the CyTOF1. When Sean Bendall optimized the labeling protocol in 2009 (resulting in the official DVS/Fluidigm protocol we all now use), one of the improvements he made was to switch to Amicon Ultra V-bottom filters. An advantage of these filters is the consistent ~20 uL residual volume, regardless of how long you spin them. With the flat-bottom filters, if you're doing multiple labeling reactions in parallel, there is a tendency to spin some filters completely dry while waiting for the slower filters to finish. This can increase variability in recovery from the filter itself. I see that Pall now has a "MicroSep" line with a consistent dead volume ("DeadStop") similar to the Amicon Ultra, so maybe those would be OK, but I wouldn't recommend the round flat-bottom ones.

ES
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ChristophS

Contributor

Posts: 21

Joined: Thu May 07, 2015 2:11 pm

Location: Switzerland

Post Tue Jun 05, 2018 12:01 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi Claude and all,

in my hands I luckily have not had intermittent results but have lost an entire batch due to an error in centrifugation:
we have an Eppendorf 5810R with 4 swing-buckets and different inserts for all kinds of tubes. Once I placed the 50k filters in the insert for 5ml tubes (they fit, but tight) and had 0% recovery.
After that I systematically kept all flow-throughs until yield was determined. Only by chance I once again made the same mistake and found almost the whole amount of Ab in the very first flow-through. The Centrifugation in the wrong insert squeezed the tube and filter so they leaked but no visible/obvious change to them. Since I take care of this, no problems anymore.

Hope this may help

Christoph
Christoph Schwärzler
Director Cytometry
Flow Cytometry Facility (https://www.cytometry.uzh.ch/en.html)
University Zürich
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Chowduck

Contributor

Posts: 29

Joined: Wed Nov 19, 2014 4:39 pm

Post Tue Jun 05, 2018 8:04 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hello Everyone,

This is most likely a membrane integrity issue that can be caused by either mechanical damage to the filter membrane from coming in contact with a pipette tip, or prolonged membrane drying that may occur, for example: in the post reduction wash steps while waiting for the polymer centrifugation to catch up. Simply not touching the membrane and adding the subsequent buffer in the protocol to the filter immediately after each centrifugation should improve recoveries.

A colleague and I did a quick test this morning to confirm this by performing the following steps:

Added 100 ug anti-mouse CD16/CD32 (clone 2.4G2) into a 50 kDa filter and topped up with PBS to 400 uL

Compared:
• Control - Pipette never touches membrane, minimum delay between centrifugations.
• Mechanical insult – lightly dragged pipette against the membrane on the way in and out of the filter.
• Drying – Pipette never touches membrane, 15-minute delay between end of centrifugation and adding PBS.

Repeated for 5 rounds of centrifugation (10min @ 12,000 rcf) and quantified each filtrate and the final retentate by nanodrop.

Recoveries were as follows:
Control (n=1): 78%
Mechanical insult (n=3): 30%, 11% and 15%
Drying (n=3): 72%, 0% and 38%

It was mildly interesting that small quantities of protein were detectable in the filtrates for the mechanical insult, indicating IgG was leaking through the filter with each centrifugation, however the drying group had no detectable protein in the filtrates. I initially expected the drying might cause membrane cracks and leaking protein but this shows it’s more likely that the prolonged drying is causing the membrane to bind more protein.
Those are my thoughts and observations on the subject. Hopefully it helps with your conjugations.

-Greg Chang
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claudechew

Participant

Posts: 10

Joined: Tue Nov 04, 2014 5:13 pm

Post Tue Jun 05, 2018 8:08 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi Greg,

That is very helpful, I am convinced that something must have changed in the quality of their membranes since a few years ago, I have always kept the same technique and washed the membranes during some of the resuspension steps, so the abrasions I'm causing by touching with the pipet tip must have been enough to cause an issue,

Thanks,
Claude
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