Ali Najafi (MRE Pharma) presents a validated LC-MS/MS MRM method for trace-level quantification of poloxamer (ploxomer) in complex biological matrices, developed to support pharmacokinetic studies and release testing for gene-therapy products. The method uses protein precipitation, solvent extraction, drying/reconstitution for pre-concentration, and external calibration in matrix. Representative validation metrics: calibration linear from 2 to 100 µg/mL, LLOQ 2 µg/mL, LOD 1 µg/mL; quadratic fit recommended. Recovery ranged 96.9–109.2%; percent accuracy ≤15%; intra- and inter-day precision (percent CV) <15% at QC levels (3.5, 35, 70 µg/mL). The talk includes background on poloxamer structure (e.g., P188: ~80 EO/27 PO; P338: ~141 EO/44 PO) and emphasizes method robustness and application to gene therapy bioanalysis.
Questions this video answers
2 µg/mL to 100 µg/mL.
2 µg/mL.
1 µg/mL.
Quadratic fit to non-zero calibrators.
96.9% to 109.2%.
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Foreign. My name is Ali Najafi. I'm a research scientist at MRE Pharma. MRE Pharma is a research and development contract laboratory based in Alameda, California. I'm very excited today to share with you a results of the method for trace level quantitative analysis of proxima in biological samples. We recently presented this work at American Society for Mass Spectrometry Conference. Currently, this method is being implemented to support a pharmacokinetic study as part of a clinical trial as well as a release testing activities for a gene therapy based product. Amphiphilic copolymers have been gaining significant attention due to their dual nature characteristics, so these polymer commonly comprise of two major moieties. As a result, these dual nature characteristics enable these polymers to be used for variety of applications including clinical gene therapy, gene delivery as well as being used as a therapeutic Sapient Poloxamer or poloronic is a synthetic amphiphilic copolymer based on hydrophilic polyethylene oxide block and hydrophobic polypropylene oxide block designed in a tri block structure. Different types of proxomer have a similar polymer backbone with two terminal ethylene oxide subunits and one central propylene oxide subunit. They have different molecular weights. For example, P188 contains 80 ethylene oxide subunit and 27 propylene oxide subunits while P338 contains 141 ethylene oxide subunit and 44 hydrophobic propylene oxide subunits. Despite the wide range of applications for ploxomer, a limited number of bioanalytical techniques has been reported in literature. Here we report robust, sensitive and specific LC Ms. Ms. MRM based methods for ploxomer quantification. Our method is mainly based on protein precipitation approach prior to LC MSMS analysis. This method has been developed to address current issues and challenges associated with analysis in trace level. Here we show a general workflow of our method so we generally start with the true biological sample containing ploxamer. We vortex and add with an extraction solvent. The extraction solvent is typically an organic solvent or a mixture of organic solvents. Afterwards we centrifuge the sample to allow protein precipitation and transfer the top layer to the new microcentrifuge tube and then we dry the top layer and reconstitute the dry extract with reconstitution solvent. The reconstitution solvent typically has the same composition as our mobile phase used for LC MSMS analysis. It is important to mention that a pre concentration step by drying and reconstituting in a smaller volume allow us to achieve a desirable sensitivity needed for trace level analysis of piloxomer. As part of a quantification activity we develop an external calibration curve by spiking P1.88 reference standard in a true biological sample. Here we show a representative calibration curve that we obtain when we develop a method for P1 ADA in a human serum. As you can see here, the linear range were found from 2mcg ML to 100 microgram per milliliter. Also a low limit of quantification and limit of detection LOD were found to be 2mcg ML and 1mcg ML, respectively. It is important to mention that the most accurate results were found to be when we apply a quadratic fit to our non zero calibrated for this method. Here we show two representative MRM chromatogram. The chromatogram on the left shows blank human serum with no spike P1.88. As you can see there is no interference at the same retention time of P188. The chromatograms on the right are three overlaid chromatograms of three independent biological replicates of P188 at low limit of quantification level. So these results show the excellent precision of the method. Also, the signal to noise of P188 peak at Lloq level allows us to do proper quantification. Also it is important to mention that the peak shape of proxomer represents a different different components of this polymer. These components have the same MRM transition but they are slightly different and resolved chromatographically due to different interactions with column stationary phase. We evaluate the percent recovery of ploxomer in biological samples so the percent recovery was calculated based on the response obtained from addition of known amount of proxomer to dried extract post extraction and compared with the response obtained from true concentration of proloxomer spike in matrix pre extraction. As you can see here, we replicate this recovery experiment by prepping three independent biological samples in three different concentration low, middle and high, all within the range of calibration curve and linear range. The percent recovery were found for all the sample from 96.9% up to 109.2% so the accuracy of quality control samples was also evaluated. The percent accuracy was calculated based on comparing the response from spike sample samples to a true concentration of the samples in all cases. In all concentration low, middle and high, the percent accuracy was found to be less than 15% which is in agreement with FDA and ICH recommendation. We also evaluate the precision or repeatability of the method. The precision of the method was evaluated by prepping three independent biological replicates of P1ADH spike sample in human serum. We selected three different levels of quality control samples including low, middle, high, 3.5, 35 and 70 microgram per milliliter, all within the linear range of the method and we calculate the precision by obtaining percent coefficients of variation percent CV for all samples and as you can see here in the table, the percent CV for all QC sample were less than 15%. We also evaluate the intermediate precision by prepping the different replicates of all QC sample in three different levels of concentrations at different date and also prepping by different analysts. And as you can see here, the percent CV for intermediate precision also found to be less than 15%. These results suggesting that the precision of the method is in agreement with the FDA and ICH recommendations. To conclude, we report a highly sensitive, robust and specific LC Ms. Ms. Based method for proxima detection in a highly complex biological sample. Here at Emory Pharma we have a team of bioanalytical scientists who are willing to tackle hard to solve problems. This is only one example, but we always look forward to bringing our customized approach to every new project. Thanks for watching.
