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Intermittent Low recoveries for MAXPAR Conjugation

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claudechew

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Post Wed Jun 13, 2018 2:11 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Just a quick reply to everyone for all your helpful suggestions! Looks like membrane integrity was indeed the culprit, repeated some of the failed batches, did the quick MilliQ test that Erin suggested and kept my pipet tip as far away as possible from the membrane and looks like that solved the problem. Back to around 70% recovery.
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fcl54643

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Location: Greater Philadelphia Area

Post Wed Jun 13, 2018 8:07 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi Claude,

Sorry for the late reply. Yes, to prime the filters, we add 200uL of R-Buffer and quick spin. Our recovery has gone back to normal since using the Amicon 30k filters and priming before adding the antibody.

My only concern with using 30k filters if the possibility that denatured antibodies could contaminate the prep, and as a result increase the background. Would anyone have some experience with or input on this?

Thank you,

Florence
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mleipold

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Location: Stanford HIMC, CA, USA

Post Wed Jun 13, 2018 8:26 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi Florence,

I'm not sure what you mean by "denatured antibodies could contaminate the prep, and as a result increase the background".

An IgG weighs about 150kDa: 2 x 50kDa (heavy chain) + 2 x 25kDa (light chain) https://www.ncbi.nlm.nih.gov/books/NBK27144/

Therefore, with complete antibody reduction down to monomeric heavy and light chains, even a 50kDa spin filter would probably retain some low amount of unconjugated Heavy chain (most filters don't have a sharp MWCO, where a 50kDa MWCO would completely remove all 50kDa species). And any Heavy chain which was conjugated to polymer (giving you background) would be >50kDa anyway.

30kDa or 50kDa filters should remove any free light chain, and any free polymer.


Mike
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ErinSimonds

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Post Fri Jun 15, 2018 11:03 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

@Greg: Thanks for sharing your very interesting test. I hadn't thought about protein drying on the membrane as a potential culprit. It is definitely something to be mindful of, as it really killed your yields -- even more than mechanical insult! This will be especially important to watch out for on the days where we are doing many spin columns in parallel. No lunch breaks after aspirating!

@Claude: Glad to hear things are working for you again!
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ssivajothi

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Posts: 31

Joined: Mon Aug 01, 2016 2:31 pm

Post Thu Jul 12, 2018 8:19 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

Hi all,

I have so far been able to avoid low recoveries by being mindful of the issues discussed here.

Sometimes, I get way more recovery than expected- 90ug (according to A280 measurement) out of 100ug input. What is going wrong here? I get signal in my run so it's not like there is no labeling. Other typical recovery is ~60ug.

The conjugation protocol from Fluidigm has two steps of 30 min washing in the polymer/metal purification step. Does everyone do both? In this case it becomes hard to coincide timing of completing both arms to proceed to conjugation step. The reduced antibody is sitting for approx 15-20 mins waiting for polymer wash to finish.

Please let me know.

Thanks you,
Santhosh
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sedejong

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Joined: Wed Mar 04, 2015 3:42 pm

Post Fri Dec 07, 2018 9:44 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

ssivajothi wrote:The conjugation protocol from Fluidigm has two steps of 30 min washing in the polymer/metal purification step. Does everyone do both? In this case it becomes hard to coincide timing of completing both arms to proceed to conjugation step. The reduced antibody is sitting for approx 15-20 mins waiting for polymer wash to finish.

I indeed do the washing steps exactly as in the protocol. After you preload the polymer with lanthanide (step 1-6), you should wait some time before you perform buffer exchange and partially reduce the antibody (step 7-12). I believe Fluidigm has tried to indicate that with the approximate time column on the left. It shows that you should only start with the antibody 30 minutes after starting with the polymer. Then your polymer and antibody will be approximately ready at the same time. To be on the safe side, make sure that that your polymer is finished earlier than antibody, as Fluidigm warns "not to allow the partially reduced antibody to remain free of the loaded polymer".
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Jahangir

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Post Wed Jan 09, 2019 11:26 am

Re: Intermittent Low recoveries for MAXPAR Conjugation

ErinSimonds wrote: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




Hi Erin,

Many thanks for your comment above, I do have a question if you don't mind.

I did what you suggested and I spun the 50kDa filter columns (4 of them in total yesterday when I conjugating some antibodies) with 400uL of MaxPar Water and measured how much water remained after 30 secs at 12,000 g. There was only around 120uL remaining in each of the 4 filter columns... so I just wanted to ask, is this level acceptable to carry on using the filters? And then I spun it down for 5 mins thereafter but there was obv around 20uL remaining at the bottom of the filter unit which I'm assuming is normal.

Many thanks in advance.

Jahangir
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eganio

Contributor

Posts: 21

Joined: Wed Nov 07, 2018 8:00 pm

Post Wed Jan 09, 2019 4:19 pm

Re: Intermittent Low recoveries for MAXPAR Conjugation

For a while in the past 2 years, I was experiencing periodic low yields from Abs from which we historically experienced good yields (~60-70%). However, I was always extremely careful not to touch the membrane while pipetting, so I suspected that something else was the culprit. I was concerned that Ab recovery at the end of the protocol might be the problem, since I did several tests where I would measure the A280 of the flow-through after each spin-down of the 50kDa filter, and found no protein in the flow-through, yet still had low recovery at the end of the protocol.

I therefore modified the protocol so that after each spin, I remove the ~20uL at the bottom of the filter and transfer to a microcentrifuge tube in order to perform the reduction and conjugation steps, as well as at the end during recovery. I also wash the sides of the filter with reduction, conjugation, or wash buffer and add it to the tube. I therefore use a fresh 50kDa filter after the reduction and conjugation steps, which means I use three 50kDa filters per conjugation. My yields have been very consistent at around 70% after modifying the protocol, and I've performed between 50 and 100 conjugations since the modification with no more low-yield problems.

This modification to the protocol costs an extra ~$6 per conjugation, but since the Abs and polymer are by far the biggest expense, it seemed worth the extra cost to prevent losing hundreds of dollars in Ab and polymer from a low-yield conjugation.
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