Post in EXT] Small scale purifications: Antibody Discussion Forum: ABRF Discussion Forums

MabselectSure (protein A based Column). We don’t see differences and in fact protein A works better for all Ig (in spite what literature says). The tip was giving to me by the company that made humanization of our Star antibody: that use mabselect for all Laura Bover, PhD (working from home during COVID-19 Cell 713-703-1951<tel:713-703-1951>) Professor Director Monoclonal Antibody Core Facility University of Texas M.D.Anderson Cancer Center Associate member of The University of Texas Graduate School of Biomedical Sciences at Houston Member of the Scientific Review Committee 2 (former Clinical Research Committee 2) Member of the Steering Committee of the Immunology Program GSBS Immunology Department/ Genomics Medicine Department 7455 Fannin St<x-apple-data-detectors://2/1> Room 1SCR4.2021 Houston- TX 77054<x-apple-data-detectors://3/0> T 713-563-3301<tel:713-563-3301> F 713-563-3276<tel:713-563-3276> <email obscured><email obscured>> In order to continue obtaining Core support in a challenging environment, it is crucial that our users acknowledge the following grants in all publications (see following text). Thank you! “We acknowledge the UT-MDACC Monoclonal Antibodies Core facility, supported in part by CCSG grant number P30 CA016672.” Sent from my iPhone On Aug 9, 2021, at 7:50 AM, Weis-Garcia, Frances/Sloan Kettering Institute <email obscured>> wrote: WARNING: This email originated from outside of MD Anderson. Please validate the sender's email address before clicking on links or attachments as they may not be safe. Yes Laura. This is exactly the stage to which I am referring. How do you purify that 100 ml of media? Over a Prot G resin using an FPLC? Be well Frances Sent on the fly from my mobile device On Aug 8, 2021, at 1:28 PM, David Blum <email obscured>> wrote: Frances: I understand. This might not be the best option, but it should be good for processing multiple samples quickly without using the FPLC. You could also batch bind with the resin you use now and spin down at a speed that doesn't deform the beads. Good luck! David On Sat, Aug 7, 2021 at 1:30 PM Weis-Garcia, Frances/Sloan Kettering Institute <email obscured>> wrote: Thanks David. The binding capacity is pretty low relative to standard resins and the beads are much more expensive. 0.6 mg rabbit IgG / ml Pierce™ Protein G Magnetic Beads while 20.0 mg human IgG / ml Cytiva Protein G Sepharose™ 4 Fast Flow Media (formerly GE LifeSciences / Formerly Pharmacia ... boy am I old) I know I want to use the FastFlow resin (or something similar) ... right now I am more looking for a process that does not involve an FPLC if I can help it. I have one but the person who has used it is retireing and this is the only thing we will use it for, so do I need it is the question. A lab (Ivo this was Pop Council) used to grow up a 100s of mls of each hybridoma then line the bottles of conditioned media up on a shelf, allowing the media to flow through a tube into a column below it and pass over the a prot G resin over night by gravity. Some how they set it up so the columns DID NOT DRY OUT. The next morning they would wash the columns by gravitty again in series and then elute them one at a time. Sounds like a great way to do a 5 to 20 at once, and I am sure I could figure this out, but before I spend time on this, was wondering if anyone else already tackled this or had a better mouse trap. Frances ZRC 1563 Office 646-888-2354 Mobile 516-967-0656

____________________________________________________________ On 8/6/21, 7:41 PM, "Antibody Discussion Forum on behalf of David Blum" < <email obscured> on behalf of <email obscured>> wrote: Have you tried mag beads? We use them for histagged proteins at small scale but there are protein coupled beads too. https://urldefense.com/v3/__https://www.thermofisher.com/order/catalog/product/88848__;!!PfbeBCCAmug!1wQ534Un_7nW4on1VYmclsK5q_UbNqiHri00ok_zzvKRc6MMxWrL3JWPqjdjH53Y$ On Fri, Aug 6, 2021 at 6:47 PM Weis-Garcia, Frances/Sloan Kettering Institute <email obscured>> wrote: Happy Friday. Do any of you produce and purify small amounts of the new MAbs you make for further characterization prior to committing to a few or one winner? If so, how do you do this. Currently we make 350 ml exhausted media (hybridoma adapted to 1% FBS prescreened for ultra-low levels of bovine IgG + 1 ng/ml IL6) and then purify it on a Prot G column using and FPLC. We get 1 – 8 mg, but I would like to scale this back and simply it if possible because we usually do not need that much. Thanks. 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