Biophysical characterization of protein reagents for batch-to-batch reproducibility

On-demand webinar

webinar-image

Summary:

The quality of protein reagents is critical to ensuring reproducibility and consistency for research. In this webinar, Deborah Moore-Lai briefly introduces a new protein product line and discusses the approach taken at Abcam to ensure batch-to-batch consistency of these proteins.

This reproducibility is validated through extensive biophysical quality control, including reversed-phase HPLC, intact mass analysis, and thermal ramp stability analysis. Using thermal stability, Abcam is able to further demonstrate batch-to-batch reproducibility using a correlation coefficient, which is generated by comparing thermal stability between individual protein batches. The full biophysical data analysis is provided for users to demonstrate Abcam’s commitment to quality and consistency, and to contribute towards successful research outcomes for its customers.

Learning objectives:

Discover and understand Abcam’s new product range, premium bioactive proteins (link to range)
Understand how Abcam ensures reproducibility and batch-to-batch consistency for proteins
Learn about the various biophysical QC methods undertaken to guarantee the highest quality product for customers

Biophysical characterization of protein reagents for batch to batch reproducibility

About the presenter:

Deborah Moore-Lai joined Abcam in 2019 to lead the new Proteins Initiative, which included building out new laboratory space and recruiting a new team of scientists with skills in protein expression, purification, and assay development. Before joining Abcam, Deborah spent 16 years working in industry in both the reagent and therapeutic spaces. For many years she led the Antibody Production team at Cell Signaling Technology. From there Deborah joined Merck Research Laboratories, where she led the team responsible for antigen and antibody generation within Biologics Discovery.

Video Transcript

00:00 - 00:17: Hi, everyone. Thank you for joining the presentation today. My name is Deborah Moore-Lai, and I am the Senior Director of Protein Development at
Abcam, located in Waltham, Massachusetts, just outside of Boston.
00:17 - 00:31: In today’s presentation, I’ll take you through some biophysical characterization of protein reagents produced at Abcam and the Waltham site,
demonstrating our batch-to-batch reproducibility of these reagents.
00:32 - 00:53: The first item I want to spend a few minutes talking about is Abcam’s commitment to quality and our supporting efforts to address the reagent
reproducibility crisis. As we all know, quality reagents are critical for ensuring reproducible research, and this is really important to Abcam to ensure that
our customers have the highest quality reagents possible.
00:54 - 01:08: The next couple slides, I’ll talk about the premium bioactive proteins, and this is a new product line at Abcam consisting of a number of growth
factors and cytokines designed for cell and gene therapy work, as well as other applications that customers may have.
01:09 - 01:22: In conjunction with that product line, we have a series of critical quality attributes that are critical for the reproducibility of these
reagents and the high-quality commitment that Abcam makes.
01:23 - 01:31: And also along those lines are analytical tools used at Abcam to assess the physical and functional characteristics of the premium bioactive
products.
01:32 - 01:35: And then in the last slide, I’ll wrap up today’s presentation.
01:36 - 01:51: On the right is a little cartoon from our Abcam website. We hosted a reproducibility workshop week in 2020 and had a number of talks, and if
you’re interested in these resources, I encourage you to go to the Abcam website.
01:52 - 02:11: Our commitment is to lower the risk of innovation and support your research efforts, and one area that we have called out are proteins, and we
believe these are reagents that require much stricter quality controls than are currently present, and we subject our in-house recombinant proteins to these
quality controls.
02:12 - 02:29: We firmly believe that quality impacts the reliability of the intended downstream applications, and our range of high-quality proteins delivers
safe and consistent reagents to support your applications, whether they’re in drug development, cell and gene therapy, or cell-based research.
02:30 - 02:37: This is some images from Abcam’s reproducibility week, and I have a paper here for Nature Communications.
02:38 - 02:55: And in this paper, what I want to call out is this desire to continue to increase the proteins and peptides, which are amongst the most widely
used research reagents, and if their quality is inadequate, what happens in the snowball effect, and how papers that are built on poor quality reagents.
02:56 - 03:12: Friedman, in the bottom right, suggests that up to 50% of preclinical experiments are irreproducible, and the cost of this is $28 billion per
annum in the U.S. alone, so as you can imagine, this impacts not just the U.S., but the global research community.
03:12 - 03:33: In the paper, Friedman recommends minimal QC tests, including SDS-PAGE or reverse-phase HPLC, to demonstrate the purity of the reagents, going
on then also to suggest an identity confirmation through LC-MS, and he suggests either intact, which is a top-down method analysis, or fingerprint, which is
essentially bottom-up.
03:34 - 03:45: And also, Abcam goes on then to validate that specific activity of the protein in the relevant functional assay, not just a binding assay, but
the functional assay.
03:46 - 04:13: This is the Friedman paper’s data, where he demonstrates this irreproducibility cost in the U.S. alone, that $28 billion that I just mentioned,
and as I said, this irreproducibility has significant downstream impacts, including the drug development pipeline, so as we know, most papers come out of
academic labs initially, and if those papers are built on faulty reagents, this snowballs.
04:14 - 04:27: And so, in the paper, they suggest an improvement in preclinical reproducibility can improve the productivity of life science research, and of
course, what we all want is to improve the speed and efficiency of therapeutic drug development.
04:28 - 04:46: And this is one protein to demonstrate this premium bioactive product line at Abcam. I have here IGF-1, where we show the data on the website,
and this supports Friedman’s suggestion that all of the critical quality attributes should be publicly available for a protein.
04:47 - 05:08: So, on the left is the screenshot of the cell-based assay for human IGF-1, and on the right is the data that Abcam provides for all proteins,
including purity, the endotoxin expression system, the UniProt accession number, the protein length, etc., and scrolling down is the sequence and the predicted
molecular weight.
05:10 - 05:14: This data package is provided for all premium proteins.
05:16 - 05:26: So, what is the premium bioactive protein line? Well, this consists of cytokines and growth factors. Right now, the product line is about 190
cytokines and growth factors.
05:27 - 05:36: And one of the quality attributes of this product line is they’re sold tag-free, and I’ll show you in a couple slides the manufacturing process
that’s undertaken to generate a tag-free protein.
05:37 - 05:52: One of the other key features is ultra-low endotoxin. So, if you use it in an assay that’s sensitive to endotoxin, such as immune cell assays,
you won’t stimulate the cells because of the presence of endotoxin. So, this product line is guaranteed to be less than 5 EU per milligram.
05:53 - 06:01: We also perform all the bioactivity testing in-house in Waltham, and it’s validated in the relevant cell-based functional assay.
06:02 - 06:08: Purity is confirmed by HPLC, and we also share with you the calculated mass determined by LC-MS.
06:09 - 06:15: We do deglycosylate the proteins just to remove that heterogeneity and then analyze on mass spec.
06:16 - 06:19: Our tolerance is less than 10 Daltons from the theoretical mass.
06:20 - 06:26: And this data package then guarantees batch-to-batch consistency and significantly improved reproducibility.
06:27 - 06:32: So, whether you buy a batch today of the protein or in 10 years, they have to meet the same quality standards.
06:33 - 06:40: And on the right is a cartoon of all of the different cytokines, many cytokines, certainly not all in the immune space.
06:43 - 06:47: We believe strongly that mammalian expression is the key to improve research outcomes.
06:48 - 06:54: On the top left is the folding process of a protein, where we go from the primary structure to the quaternary folded structure.
06:55 - 07:01: And as this audience knows, that properly folded structure is required for optimal bioactivity.
07:02 - 07:06: Without a properly folded protein, it’s difficult to assess the activity of that protein.
07:07 - 07:15: The bottom left is a reverse phase image, and in a few minutes, I will show you some examples of proteins that have failed reverse phase.
07:15 - 07:23: And the lab here in Waltham fails them for the quality reasons, and so our commitment to ensure high-quality reagents.
07:24 - 07:30: On the top right, of course, these proteins are produced exclusively from mammalian cells.
07:31 - 07:40: And of course, this is the eukaryotic cell with the ability to fully modify that protein with post-translational modifications that are relevant
for function.
07:41 - 07:45: And on the bottom right is an example of the intact mass analysis.
07:50 - 07:53: As I said, post-translational modifications are critical for structure and function.
07:54 - 07:58: And there’s an enormous amount of modifications that happen to proteins once they’re produced.
07:59 - 08:05: So we go from the transcriptome to the proteome, and then the final protein species that carry all of these different modifications.
08:05 - 08:07: And on the top right are the most common.
08:08 - 08:15: This list is certainly not complete, but these are the most common modifications that we would find in a eukaryotic system.
08:16 - 08:22: The strongest modification that we have coming out of the mammalian for our purposes, of course, is glycosylation.
08:23 - 08:28: So we want those proteins to be glycosylated to improve their function and also enhance their stability.
08:29 - 08:34: The production workflow for this product line is about a two-week process.
08:35 - 08:37: We start with the mammalian cell expression system.
08:38 - 08:42: And the constructs are designed, the proteins are fully designed in the Waltham lab.
08:43 - 08:47: And we have the gene synthesized and inserted into the vector.
08:48 - 08:52: The initial construct does carry the tags.
08:53 - 09:00: And then when the protein is expressed in step three through mammalian transfection, it is expressed with the tag.
09:01 - 09:04: And then in step four, the proteins are purified using that tag.
09:05 - 09:10: But it’s about a four-day purification process from the time they’re initially bound to the column,
09:11 - 09:14: and then they’re cleaved to remove the tags and then re-purified.
09:15 - 09:19: And then re-purified to get an ion exchange or gel filtration as needed to improve the purity.
09:20 - 09:24: Step six is another unique quality for this product line.
09:25 - 09:29: The proteins are lyophilized, and our current size is 10 or 50 microgram vial sizes.
09:30 - 09:33: And what this means for you is long-term stability on the shelf.
09:34 - 09:39: And also, when you want to use the protein, you can resuspend to the concentration that you desire for your assay.
09:40 - 09:44: After lyophilization, the proteins go through this extensive quality control,
09:44 - 09:50: including the HPLC, the LC-MS, the endotoxin, and the SDS-PAGE, and the cell-based assay.
09:53 - 09:56: Just to give you another in-depth window into protein production at Abcam,
09:57 - 10:02: we do have several sites around the globe that do protein production, and we all use the same methods.
10:03 - 10:05: So it starts with protein design on the left.
10:06 - 10:12: In the case of an antibody, we want to identify the best sequence to identify the best antibody binder.
10:13 - 10:16: So we want the right construct and the right expression system.
10:17 - 10:19: Then we screen. So we screen in small scale.
10:20 - 10:28: Depending on the scale, it can be anywhere from a mil to 100 mils to find the best expression conditions for success.
10:29 - 10:33: Then we go to protein production, and then quality control, and finally that protein delivery.
10:34 - 10:37: And what you see on the bottom right is a customer-facing vial,
10:38 - 10:41: and we will either generate these lyophilized, or we can also generate liquids.
10:42 - 10:49: Again, improving experimental reproducibility through in-depth protein analytics.
10:50 - 10:55: And what we have here are the protein analytics for human TNF-alpha 259410,
10:56 - 10:57: where we have the activity assay.
10:58 - 11:04: The ED50 there is presented and is determined by dose dependence, apoptosis of L929 cells.
11:05 - 11:11: You can see the SDS-PAGE, the reverse phase, greater than 95% purity, and the LC-MS data.
11:13 - 11:17: So in the next couple of slides, what I want to take you through are some real-world evidence.
11:18 - 11:22: This is real data from the Waltham Lab showing you what passes our quality control
11:23 - 11:28: and what does not pass our quality control to, again, to support our commitment at Abcam to high-quality reagents.
11:29 - 11:33: So again, our tolerance for reverse phase HPLC is a purity greater than 95%.
11:34 - 11:39: So the top left graph, you can see this really beautiful, sharp, symmetrical peak.
11:39 - 11:42: No evidence of a shoulder on the left or the right of the peak.
11:43 - 11:48: Of course, the shoulders would indicate additional species in the sample, whether it was degradation or aggregates.
11:49 - 11:53: So this is the kind of purity data that qualifies a protein to be a premium protein.
11:54 - 11:57: Protein X on the bottom right, well, it is a nice, sharp peak.
11:58 - 11:59: You can see that shoulder and that tailing effect.
12:00 - 12:04: So of course, this is suggestive of impurities and aggregates in that protein preparation.
12:05 - 12:10: And unfortunately, in many cases, they simply can’t be removed, even through a multi-step purification process.
12:11 - 12:13: So this protein is not released as a premium protein.
12:14 - 12:16: It did fail that QC criteria.
12:19 - 12:25: Demonstrating some actual LC-MS data, and again, our tolerance from the calculated mass, the theoretical mass is 10 Daltons.
12:26 - 12:31: And as I mentioned earlier in the talk, we do deglycosylate with all the N-glycans come off
12:31 - 12:34: and the simple O-linked glycans will come off with our enzyme.
12:35 - 12:42: And so on the left, you can see it matches the mass perfectly, the 17,410 Daltons.
12:43 - 12:45: And what I have there is the sequence below.
12:46 - 12:48: And I think this is important because the protein on the right,
12:49 - 12:52: the yellow highlighting the glycosylation motifs, just so we can take a look at that.
12:53 - 12:56: So proteins that aren’t carrying any glycosylation like the TNF-alpha,
12:57 - 12:59: they are typically a really beautiful peak on LC-MS.
13:00 - 13:04: The protein on the right, sometimes you can see an additional mass
13:05 - 13:07: due to those additional glycans that are left on there.
13:08 - 13:10: But in this case, this protein did fail.
13:11 - 13:14: We can see a significant cleavage effect in the protein.
13:15 - 13:19: And so because it was significantly greater than 10 Daltons due to this truncation,
13:20 - 13:22: we did fail this as a premium protein.
13:23 - 13:27: Again, thorough QC does require multiple analyses to determine the purity.
13:28 - 13:31: So we consider QC a holistic approach.
13:32 - 13:34: And so when you start with the SDS-PAGE gel,
13:35 - 13:37: of course, SDS-PAGE gels are quickly generated.
13:38 - 13:39: Some of the other assays can take a day or two.
13:40 - 13:45: So in this protein, it’s a beautiful greater than 95% purity on the SDS-PAGE gel.
13:46 - 13:48: When we ran the LC-MS, the top right,
13:48 - 13:50: we saw a lysine truncation, which is not unusual.
13:51 - 13:53: Terminal lysines will tend to truncate.
13:54 - 13:56: So we were able to explain that truncation.
13:57 - 14:02: And looking at the bottom, we call that a greater than 95% purity on HPLC.
14:03 - 14:06: And then once these items pass, we will take them on to the cell-based assay.
14:07 - 14:10: So this assay for FGF-19, you can see passed.
14:11 - 14:16: We had a dose-dependent response with proliferation of the NIH cell-based assay.
14:16 - 14:18: With proliferation of the NIH C23 cells.
14:19 - 14:24: So this protein fully passed all requirements for a premium bioactive protein.
14:26 - 14:30: In contrast, this PTX3 human protein was a bit more challenging.
14:31 - 14:33: And again, what you can see, the SDS-PAGE gel looks pretty clean.
14:34 - 14:36: We’re not seeing additional bands or masses on the gel.
14:37 - 14:39: And when we took it to LC-MS,
14:40 - 14:43: we saw a lot of additional masses that we were not able to explain.
14:44 - 14:47: And it’s not suggesting that there’s impurities in there,
14:48 - 14:50: but there’s definitely cleavage events that we don’t understand
14:51 - 14:52: that are happening during production.
14:53 - 14:56: And this is also further substantiated by this reverse phase data at the bottom,
14:57 - 14:59: which is kind of a nasty peak.
15:00 - 15:03: So this failed QC. We would not release this as a premium protein.
15:06 - 15:07: Another protein to give you an example.
15:08 - 15:10: Here, the SDS-PAGE was a little bit questionable.
15:10 - 15:13: We can see that doublet, although often you can explain a doublet
15:14 - 15:16: through glycosylation, which if we look at the top right,
15:17 - 15:18: you can see the yellow highlight.
15:19 - 15:21: There is a glycosylation site on VEGF-A.
15:22 - 15:24: When we looked at the LC-MS,
15:25 - 15:27: then we were able to see those glycans, right?
15:28 - 15:30: They’re annotated there on the LC-MS.
15:31 - 15:35: And we had a 162 Dalton cleavage event due to an arginine cleavage,
15:36 - 15:37: again, which we were able to explain.
15:37 - 15:39: And often biologically, these arginines,
15:40 - 15:42: we see terminal arginines will cleave during production.
15:43 - 15:46: So because we were able to explain the biology of the protein,
15:47 - 15:48: we did pass QC.
15:49 - 15:51: This particular protein, I don’t have the cell-based assay data.
15:52 - 15:53: That is still in progress.
15:55 - 15:58: So available for all proteins, for any customer that requests,
15:59 - 16:01: we have this full certificate of analysis.
16:02 - 16:05: And you can see on the left, we have very detailed information
16:05 - 16:07: and all of the required QC data that is generated
16:08 - 16:10: for each batch of protein that is produced.
16:11 - 16:15: And on the right, you can see the sign-offs from myself and from Quality.
16:19 - 16:21: We were also curious about the quality of our proteins
16:22 - 16:23: against some of our competitors.
16:24 - 16:25: So we engaged with a third-party CRO
16:26 - 16:27: to perform head-to-head data comparison.
16:28 - 16:30: And the CRO confirmed our premium bioactive proteins
16:31 - 16:34: are of equivalent or higher potency to our competitors in every case.
16:35 - 16:37: And you can see the EC50s at the bottom
16:38 - 16:39: with the Abcam protein in the blue
16:40 - 16:42: and the competitors in the green and the red.
16:47 - 16:51: A continuation of that data to further show the purity of the proteins.
16:52 - 16:54: So here we have the proteins, the three proteins,
16:55 - 16:57: and we have multiple batch repeats.
16:58 - 17:00: So this goal was to show batch-to-batch consistency.
17:01 - 17:03: Batches were produced in separate days,
17:03 - 17:04: separate operators.
17:05 - 17:07: And the reverse phase here, this is the retention time.
17:08 - 17:11: In the case of TNF-alpha, 6.1 minutes, and the mass.
17:12 - 17:14: And you can see how these batch-to-batch reproducibility
17:15 - 17:17: is very consistent between these three proteins
17:18 - 17:19: and three separate batches.
17:20 - 17:21: And on the bottom is that cell-based assay data.
17:22 - 17:25: Again, these are proteins that were entirely produced within Abcam.
17:26 - 17:28: But this consistency of data demonstrates our commitment
17:29 - 17:30: to reproducibility.
17:33 - 17:36: So in the last few minutes of my talk,
17:37 - 17:39: I want to talk about thermostability analysis and protein stability.
17:40 - 17:43: And one of my favorite proteins is TNF-alpha human,
17:44 - 17:46: and that is the cartoon here of TNF-alpha.
17:50 - 17:53: As this audience knows, protein stability is critical
17:54 - 17:56: for every functionality of a protein.
17:57 - 18:00: And misfolded proteins in vivo have catastrophic effects
18:00 - 18:02: and can play prominent roles in disease states.
18:03 - 18:05: And on the right are several diseases
18:06 - 18:07: that I’m sure this audience is quite familiar with
18:08 - 18:10: that are a result of protein misfolding,
18:11 - 18:13: from Alzheimer’s, Parkinson’s, Mad Cow,
18:14 - 18:17: and transthyretin amyloidosis.
18:19 - 18:24: And just to further explain the level of protein organization
18:25 - 18:26: and how that affects overall function,
18:27 - 18:29: so we go from the primary to the secondary, tertiary,
18:30 - 18:33: and quaternary structure to as the protein starts to fold
18:34 - 18:36: inside the cell, and it’s that quaternary structure
18:37 - 18:39: that’s critical for the stability.
18:41 - 18:44: So on the right, I have some quotes
18:45 - 18:46: from the AlphaFold protein structure database
18:47 - 18:49: that you can take a moment to read.
18:50 - 18:52: But protein stability on the left can be thought of
18:53 - 18:54: as a thermodynamic preference.
18:54 - 18:56: So it wants to achieve a folded state
18:57 - 18:58: and maintain that state.
18:59 - 19:00: And this is critical for protein function
19:01 - 19:02: and, of course, prevents accumulation
19:03 - 19:04: of unfolded and misfolded protein forms
19:05 - 19:06: in the case of some of those diseases
19:07 - 19:08: on the previous slide.
19:09 - 19:11: One measurement that people use for protein stability
19:12 - 19:13: is the melting temperature.
19:14 - 19:16: So if you think about as a protein starts to heat,
19:17 - 19:19: and as you heat it and it starts to unfold,
19:20 - 19:21: it starts to aggregate.
19:22 - 19:23: So in the next slide, I’ll show you
19:24 - 19:26: how we measure protein as it starts to unfold
19:27 - 19:29: and then as it starts to aggregate.
19:30 - 19:31: Stability information, of course,
19:32 - 19:34: this is the holy grail that we’re all trying to understand,
19:35 - 19:36: is what dictates protein folding.
19:37 - 19:39: We do know it’s encoded in the protein structure
19:40 - 19:41: determined by the primary sequence.
19:42 - 19:43: And this has stood as a challenge
19:44 - 19:45: for more than 50 years now.
19:47 - 19:48: So in terms of protein stability,
19:49 - 19:51: the empirical determination of the melting temperature,
19:51 - 19:53: we can screen this using melting temperature analysis.
19:54 - 19:56: And this can be useful for many things,
19:57 - 19:58: including identifying ligand and buffer conditions.
19:59 - 20:01: So you have proteins in different conditions,
20:02 - 20:03: and then you can assess their stability
20:04 - 20:06: and their melting temperature.
20:09 - 20:10: This is important for purification,
20:11 - 20:12: crystallization, and functional characterization.
20:13 - 20:14: And so this destabilization and melting
20:15 - 20:16: is measured as those hydrophobic sites.
20:17 - 20:18: So again, as the protein starts to unfold
20:19 - 20:20: and become exposed,
20:21 - 20:23: and you’ve treated the protein with a fluorescent dye
20:24 - 20:26: that binds to certain regions and then starts to fluoresce.
20:27 - 20:28: And the melting temperature is identified
20:29 - 20:31: at the point where 50% of the protein is unfolded.
20:32 - 20:33: So the chart on the left,
20:34 - 20:35: you can see the temperature on the x-axis
20:36 - 20:37: and the fluorescence on the y.
20:38 - 20:39: And as the temperature starts to go up
20:40 - 20:41: and the protein starts to unfold,
20:42 - 20:43: and then that dye can actually get inside the protein
20:44 - 20:45: and starts to fluoresce.
20:46 - 20:47: And then when the protein is fully denatured,
20:48 - 20:49: then there’s maximum dye binding.
20:50 - 20:51: And then finally, this protein aggregation
20:52 - 20:53: as it starts to fold upon itself.
20:56 - 20:58: One instrument that we use in the Waltham site
20:59 - 21:01: is the UNcle from Unchained Labs.
21:02 - 21:04: And the UNcle does a variety of analytical techniques.
21:05 - 21:08: It will do DLS, DSF, melting temperature,
21:09 - 21:11: and it does it in a high-throughput,
21:12 - 21:13: automated fashion.
21:14 - 21:16: And for us, it’s desirable to have an orthogonal method
21:16 - 21:17: to query protein stability.
21:18 - 21:19: And we came at this
21:20 - 21:21: because we wanted this additional method
21:22 - 21:23: to correlate physical structure
21:24 - 21:25: as a surrogate for functional studies
21:26 - 21:27: and to confirm our functional studies
21:28 - 21:29: with the physical structure of the protein
21:30 - 21:31: to understand the folded state.
21:32 - 21:33: So we employ this instrument
21:34 - 21:35: to assess the thermal stability.
21:36 - 21:37: And we compare subsequent batches of proteins
21:38 - 21:39: against each other.
21:40 - 21:41: So a batch that’s produced today
21:42 - 21:44: is compared in six months on the UNcle.
21:45 - 21:47: We use a slightly different metric,
21:48 - 21:50: the Thermal Stability Correlation Coefficient, TSTC.
21:52 - 21:54: And we then establish a correlation coefficient
21:55 - 21:56: between subsequent batches of protein.
21:57 - 21:58: So the correlation coefficient
21:59 - 22:00: of the melting temperature between two proteins,
22:01 - 22:02: and in this case, as I said,
22:03 - 22:05: between the same protein produced in two separate batches.
22:06 - 22:07: And there’s two contributions
22:08 - 22:10: that determine the thermal properties of a protein.
22:11 - 22:13: I guess the thermodynamic stability
22:14 - 22:16: which is defined as the difference in free energy
22:17 - 22:18: between the folded and unfolded states
22:19 - 22:21: and the melting temperature of the protein.
22:23 - 22:25: So this is some actual raw data from the lab
22:26 - 22:27: where we had a protein,
22:28 - 22:29: three separate batches of a protein
22:30 - 22:31: were analyzed in triplicate.
22:32 - 22:34: And then we determined the correlation coefficient
22:35 - 22:36: between the individual batches.
22:37 - 22:38: And you can see the data at the top.
22:39 - 22:41: Those are the three individual correlation coefficients,
22:42 - 22:43: all greater than 0.99.
22:45 - 22:46: And so based on this,
22:47 - 22:49: we concluded that the subsequent batches of protein
22:50 - 22:51: were identical
22:52 - 22:53: in terms of their analysis
22:54 - 22:55: in the thermal stability curve,
22:56 - 22:57: which then when we correlated that
22:58 - 22:59: to the functional testing,
23:00 - 23:01: we satisfied ourselves that this is an orthogonal method
23:02 - 23:03: to assess protein quality
23:04 - 23:05: against functional testing.
23:10 - 23:11: So in wrap-up,
23:11 - 23:13: Abcam is committed to improving reagent reproducibility
23:14 - 23:16: and minimizing batch-to-batch variability.
23:17 - 23:18: And I shared with you data
23:19 - 23:20: that’s generated for every batch of proteins
23:21 - 23:22: in my presentation today.
23:23 - 23:25: We’ve introduced this class-leading product line,
23:26 - 23:27: the premium bioactive proteins,
23:28 - 23:29: where we provide full biophysical characterization
23:30 - 23:31: for each protein.
23:32 - 23:33: The internal team in Waltham
23:34 - 23:35: has an in-depth knowledge of each protein structure
23:36 - 23:37: and function.
23:38 - 23:39: If you ever have a question,
23:39 - 23:40: please reach out to technical support
23:41 - 23:42: and you will come straight to us
23:43 - 23:44: and we are happy to talk to you.
23:45 - 23:46: And we’re also happy to talk to you
23:47 - 23:48: about the cell-based assays that we perform.
23:49 - 23:50: So please reach out to technical support at Abcam.
23:51 - 23:52: We provide this full biophysical profile
23:53 - 23:54: for each batch of proteins.
23:55 - 23:56: Again, going back to the paper that I referenced,
23:57 - 23:58: where there’s some minimal QC data
23:59 - 24:00: that should be provided for all proteins.
24:01 - 24:02: In our case, the gel, the reverse phase,
24:03 - 24:04: LC-MS, thermal stability,
24:05 - 24:06: and functional study data.
24:07 - 24:08: And all this culminates
24:09 - 24:10: in our core belief
24:11 - 24:12: that we have the highest quality proteins in the market.
24:13 - 24:14: They’re correctly folded
24:15 - 24:16: and they carry the mammalian
24:17 - 24:18: post-translational modifications
24:19 - 24:20: because of their production in 293 cells.
24:21 - 24:22: We provide the biophysical characterization
24:23 - 24:24: and the functional activity
24:25 - 24:26: confirmed in the relevant assay.
24:27 - 24:28: In closing, Abcam also offers custom solutions
24:29 - 24:30: and we’re always looking to work directly with you.
24:31 - 24:32: The lab in Waltham
24:33 - 24:34: has produced many custom proteins for customers,
24:35 - 24:36: some really interesting proteins.
24:37 - 24:38: And so please reach out to us directly
24:39 - 24:40: and we’re happy to talk to you
24:41 - 24:42: about what your custom project might look like
24:43 - 24:44: to develop new reagents
24:45 - 24:46: and to meet your research demands.
24:47 - 24:48: Thank you so much
24:49 - 24:50: and I’m happy to answer any questions.
24:50 - 24:51: Thank you.

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