Prazitha Pandey describes an LC-MS/MS method that quantifies all six monoclonal antibodies in a cocktail with one assay instead of six separate ELISAs. The approach uses trypsin digestion to create unique peptide signatures for each antibody, then optimizes mass spectrometry transitions to measure them simultaneously with high specificity and sensitivity.
Questions this video answers
Prazitha Pandey, Assistant Director Chemistry at Emory Pharma.
Trypsin (proteolyze using trypsin).
5 to 25 amino acids.
1 to 1500 microgram per milliliter.
0.1 to 10.3 microgram per milliliter (at signal-to-noise = 10).
Chapters
Foreign. Hello everyone, my name is Prazitha Pandey and I'm Assistant Director Chemistry at Emory Pharma. Today I'm here to discuss LC Ms. Ms. Method development to quantitate each antibody from a six antibody cocktail in human serum. Traditionally this kind of work is done using ELISA 1 which would require six independent ELISA assays. But whereas I'm here to present today some advantages of using LC Ms. Ms. For this kind of workflow and also walk you through some stepwise approach to get a single workflow. Before we move on to the LC Ms. Ms. Method development, let's discuss some of the advantages of using LC Ms. Ms. Method. As discussed earlier, ELISA would require six different independent assays to do such kind of work, whereas LC Ms. Ms. Is able to do this all in a single run which would require a single method that would be able to quantitate all six antibodies. LC Ms. Ms. Would require fewer critical reagents as compared to multiple reagents needed for elisa. Further, LC Ms. Ms. Is less prone to matrix interference as compared to ELISA which can be affected by multiple components within the sample leading to inaccurate results. Now that we have discussed the advantages, let's see the scope of work and let's do a stepwise approach to have that workflow established to quantitate all six antibodies in a single so the goal would be to develop LC MRM method which would be very specific for each of these antibodies using in human serum matrix. Starting with in silico analysis where we would generate a theoretical mass spectrometry data and also find a unique peptide against a human serum matrix and against the five other antibodies. Then we move on to the LCMRM method where we experimentally determine what is the precursor ions, what are the possible product ions and what is in optimal MRM transition to quantitative each of these antibodies in the drug product and lastly do some method evaluation where we look into the specificity, selectivity, sensitivity, linearity range and accuracy of this method starting with in silicoanalysis. This would require your protein sequence to be analyzed in multiple platforms such as whether it's NCBI peptide or SKYLINE to generate that theoretical mass spectrometry data and hence determine the unique peptide against human proteome and also unique peptide against the five other antibodies that is within the drug product. This image here shows you the overall workflow which is using a bottom up approach. Starting with the six proteins we would then proteolyze using trypsin, generate peptides coming from each of these proteins, do LC Ms. Ms. Analysis on it and generate MRM transitions for quantitation purposes. While deciding on surrogate peptides, there are some key consideration that needs to be be kept. And as I mentioned earlier, it's very important to discuss what are the peptide that is unique against human proteome database as well as unique against five other antibodies. The other important thing is also to look at peptide length because chromatographically you want to have a peptide length between 5 to 25amino acids so that it doesn't elute in your solvent front or cause any peak tailing. Further, you want your sequence to not have any sites prone to missed cleavages and also prone to modifications such as oxidation and deamidations. Once you have determined that sequence in silico, we have to make sure that those peptides are also chromatographically observed in the mass spectrometry data. So, starting with LC Ms. Method development and prior to moving LC Ms. Ms. Method development, it is very important to look at those precursor ions. So in this workflow you can see that there are six proteins where we proteolyze using trypsin, generate the peptides, conduct LC Ms. Analysis on it. Now, on this step, it's very important also to look at the protein to trypsin ratio and also the incubation time so that you get the precursor ions with highest intensity. Once you have generated the precursor ions in mass spectrometry, it's very important to look at what precursor you want to proceed with to conduct Ms. Ms. Analysis. In this example here on top, you can see a peptide of interest and you can see at the bottom that there are three different charges, singly, doubly and triply. You can clearly see here that the doubly actually has the highest intensity and as a result would be taken forward to conduct Ms. Ms. Analysis. Now moving on to LC Ms. Ms. Analysis from LC Ms. Analysis. So this would make sure that we have a product ion coming from the precursor ions and also have that optimal Mr. Transition that would be able to quantitate all six antibodies in this drug product. So for that reason and for that purpose, we optimize various mass spectrometry conditions here, such as fragmentary voltage collision, energy, gas flow rate and cell accelerator voltage and confirm the mass M over Z of the product ion that is coming out of this precursor ion and determine the suitable MRM transitions to generate a single workflow. In this chromatogram you can see that MRM transitions that is being generated from all six antibodies. The reason you are seeing multiple Peaks in this chromatogram is because we had chosen two to three peptide for antibodies and two transitions per peptide. But it's very important to note that all of this was actually done using a single workflow. Once you have a method, it's very important to determine some key parameters such as specificity and selectivity, linearity and range. So couple of my slides now would show you some of the parameters we had developed for this particular work. Some of the key parameters that was evaluated starting with specificity and selectivity. In this example here you can see peptide of interest chromatogram being overlaid with human serum and human serum and other 5 antibodies. As you can see in the figure at 32.752 you see a peak eluting from peptide of interest. But there is no interference from human serum and human serum and other five antibodies confirming the selectivity and specificity of this method. Matrix interference Another key parameters that was evaluated for this purpose, we compared the drug product in buffer versus drug product in human serum. As a result, as you can see here that we did see some level of matrix interference and for all six antibodies it ranged somewhere from 16.7% to 40%. Moving on to the linearity range, this linearity end range, the calibration curve was created from the drug product which means that all six antibodies were diluted to make that calibration range. For this purpose we created a range from 1 to 1500 microgram per milliliter. And for this analysis we were able to get R square more than 0.9. And lastly sensitivity which is key to any bioanalysis. And for this purpose we looked at the sensitivity which means looking at the limit of quantitation at signal to noise 10 and we were able to get the limit of quantitation ranging from 0.1 microgram per mil to 10.3 microgram per mil ranging for all six antibodies. So to summarize, we were able to generate a LCMRM Ms. Method to quantitate all six antibodies using a single workflow. This can be a very valuable approach for bioanalysis of biotherapeutics, specifically when there are multiple APIs involved. And this can be a cost effective and a time efficient method and approach in bioanalysis. To discuss your projects with one of our scientists, please contact@bdmeryformant.com Sam.
