Tag: 18LiyaC

We spent our final day at Tergus Pharma learning from a member of the data analysis team named Praneetha. She helped us understand some more details of the operations of HPLC, discussing different types of mobile phases, two examples of stationary phases used in the columns, and functions such as the maximum pressure limit that protect the machine from possible issues with blockages. She emphasized the importance of system suitability: checking the accuracy, precision, and tailing factor of the graphs, among other guidelines, in order to ensure that the experiment can be performed successfully and repeated reliably. She also pointed out that every step and mistake made when writing in her project notebook had to be carefully documented and explained with footnotes. Then, she showed us how to prepare a standard using a placebo gel. While waiting to use a balance, we saw another scientist calibrate it by massing known weights and comparing them with a range of accepted values that fell within 1% of the actual weight. Once Praneetha had massed out the sample of placebo gel, she diluted it and used a vortex machine and sonicator to homogenize the mixture.

After lunch, Praneetha showed us some examples of HPLC graphs, which are used to check the amount of active ingredient present in a drug. As the day came to an end, she and Ravi also answered a few more questions for us. They explained that gas chromatography differs from the liquid chromatography that we have been seeing for the past few days in that the sample volatilizes while passing through coils to allow for more surface area and time. They also discussed different types of HPLC columns and the methods used to test drugs for stability over time. Finally, we said goodbye to Duffy and had a chance to meet the CEO of Tergus, Dr. Nalamothu, who had been away at a conference all week. The work I saw over the past few days definitely exposed me to different aspects of pharmaceuticals and the drug development process that I had not known about beforehand, and I’m glad to have had this valuable experience.

Today, we shadowed a few scientists at in vitro release testing (IVRT). To start off, Craig, the head of the IVRT team, talked us through some of the experiments performed and methods used by IVRT, including different dosing techniques and strengths of drugs and how they impact the release rates. Then, Akeem demonstrated a test using vertical diffusion cells. The setup was similar to what we saw yesterday, simulated the properties of skin with a synthetic membrane rather than using actual skin, requiring a smaller time frame for each experiment. In order to determine the release medium that would result in an optimal dose-release ratio, Akeem tested two different solutions: a 50:50 mixture of water and methanol and a 70:30 ratio of water and ethanol. Rather than applying the cream with a pipet as the scientists in IVPT did, he added a constant volume of the drug to each cell by filling the hole in a washer-shaped wafer placed on top of the membrane.

After lunch, we talked with Meredith, who explained some of the functions of HPLC machines and what she looks for on the chromatographs. Then, Ms. Allen paid us a visit, and we discussed our experiences over the past two weeks. Finally, we met with Craig again; he discussed some more of the FDA regulations and guidelines that have to be followed when testing new proposed drugs, as well as the different types of tests performed on the drugs, some of which can take years to complete.

We spent our second day at Tergus Pharma with in vitro permeation testing (IVPT). Monica and Brandon, the two scientists, tested the penetrating ability of drugs on cadaver skin samples by using vertical diffusion cells. Each small glass cell was filled with a mixture of water and receiving medium to simulate the properties of blood, then covered with a small piece of preserved skin. There were three drugs analyzed today: an original drug already on the market, a proposed generic version of said drug, and a challenge sample mean to create differing results from the other two. Small drops of the liquified drugs were pipetted onto the skin and spread out with a spatula. Samples of the solutions below the skin were to be taken every 4 hours over a 72-hour period and analyzed using HPLC and mass spectrometry to determine the effectiveness of the drugs. Monica explained and demonstrated some of the guidelines of GLP (good lab practice); she and Brandon recorded data for each step of the procedure to ensure that the experiment could be successfully replicated if necessary, and an auditor from quality assurance monitored the dosing process.

Later in the afternoon, we shadowed Lynn, another member of the IVPT team. From what I understand, she was calibrating a mass spectrometer, varying the concentration of the added substance to optimize the peak height and width displayed by the detector. Overall, though I didn’t get to do any hands-on experimenting (with good reason), today’s experiences were a fascinating look into the details of the drug development process.

This week, we’ll be interning at Tergus Pharma, a company focusing on the development of topical treatments. After arriving, we sat down with Duffy McDonald and Wendy Ward from the Human Resources Department; they helped introduce us to Tergus by providing some background on the history of the company as well as on their own journeys to their current positions. Then, we went on a tour of all the labs and offices in the building with Chris and Michael, who work with operations and facilities; a few of the labs included equipment that we also encountered last week at Integrated Pain Solutions, including HPLC and mass spectrometers. Afterwards, we were given a presentation by Avinash, who works on the business side of the company. We learned about the drug development cycle, from discovery and development to FDA post-market safety monitoring, along with exploring the different aspects of testing treatments during the development stage.

We will be learning more about these steps of drug development in chronological order throughout this week. This afternoon, Srikanth, one of the scientists, brought us to the labs to teach us about formulation. He first demonstrated how to create a hydrogel by combining water, sorbitol, sodium hydroxide as a neutralizer, glycerin, and the gelling agent carbopol 980 in a small mechanical stirrer. While waiting for the ingredients to mix together, he also discussed creating creams and testing mixtures with HPLC, microscopes, texture analyzers, viscometers, pH meters and rheometers. Altogether, we covered a lot of information today, and I’m looking forward to the week to come!

To start off the day, Sean showed us how he prepared a sample of viscous, almost pure CBD oil (about 98% CBD and 2% THC) for analysis with the HPLC and tandem mass spectrometry. The THC percentage was higher than permissible by the state law; he diluted the sample by pipetting in isopropanol. Then, we shadowed Ashleigh as she set up samples of urine for confirmation testing with HPLC; she included small volumes of the samples, a clean control, and two samples containing all of the substances being tested for. After lunch, we helped Stephanie label bottles of hemp oil in Integrated Hemp Solutions and continued to shadow Sean and Ashleigh.

Overall, I thoroughly enjoyed being able to intern in such a relaxed yet intriguing environment; I’m grateful to have heard about the scientists’ personal experiences and seen some applications of the knowledge I gained in classes and clubs at school though learning about the lab and hemp aspects of the business.

This morning, we continued shadowing Sean as he analyzed the samples of oven-dried hemp flowers and CBD oil. He showed us the collected data displayed on his computer and talked us through the process of creating a report for the concentrations and percentages by mass of the components of each sample. We then went back to the lab to clean the two mass spectrometers, a procedure repeated on a weekly basis. After taking apart each mass spectrometer, Sean wiped down the larger parts, first with pure H2O and then with isopropanol. He also placed the smaller metal parts used to conduct ions into a graduated cylinder filled with water; this graduated cylinder was then moved into a sonicator, a machine that applies ultrasonic frequencies that cause the parts to vibrate and dislodge any trapped ions or impurities.

In the afternoon, Krystle showed us how she performs screening tests for urine samples to determine whether they contain certain drugs and ensure that they have not been tampered with or diluted. The Enzyme Multiplied Immunoassay Technique (EMIT) detects enzyme activity in a sample in order to measure the concentration of drugs. Point of Care Testing (POCT) cups with strips of nitrocellulose membrane lining their walls are filled with urine samples; the number of lines appearing on the strips indicates whether a sample tests positive or negative for each drug. Krystle also emphasized the importance of conducting confirmation testing using HPLC: immunoassay testing cannot differentiate between compounds with similar molecular structures, such as CBD and THC, while confirmation testing can more accurately determine the compounds present.

Today, we spent the morning assisting Sean, one of the scientists, in the Integrated Lab Solutions area of the clinic. Sean gave us a quick introduction to high-pressure liquid chromatography (HPLC) and tandem mass spectroscopy, two methods employed by the equipment in the lab. HPLC separates and then determines the identities and concentrations of the components of a substance by comparing the retention times (time needed to detect molecule) and peak heights detected with those of known molecules. Tandem mass spectroscopy ionizes a sample, breaks those ions down into fragments, and then detects and identifies specific product ions. We used these techniques to analyze seven components of hemp, including the cannabinoids CBD, THC, CBDA, and THCA, thus ensuring that the hemp products contained acceptable levels of each substance. We helped Sean prepare samples of dried hemp flower and CBD oil to analyze in the HPLC and tandem mass spectroscopy machines by pipetting different amounts of each substance into small vials and diluting them with isopropanol.

After lunch, we conducted further research on HPLC and tandem mass spectroscopy independently while waiting for the results of the sample analysis. We went more in-depth to solidify our understandings of the two methods, learning about the different types of HPLC (normal phase and reversed phase) and its detection methods (UV absorption, fluorescence, etc.), along with the stages and applications of tandem mass spectroscopy.

For the first week of the work experience program, Hern and I are working at the Integrated Pain, Lab, and Hemp Solutions center in Sanford. After receiving a brief tour of the facilities, we conducted some independent research on cannabidiol (CBD) based on a set of guidelines provided by an employee. CBD, a cannabis compound derived from hemp or marijuana plants, relieves pain, inflammation, and other ailments, and is used in conjunction with surgical procedures and movement therapy at Integrated Pain Solutions. We learned about its effects, mechanisms, and legality, along with exploring liquid and gas chromatography and mass spectroscopy. In the afternoon, we had the opportunity to see the process behind distributing the hemp oil; we helped dispense, bottle, and package a mixture of CBD oil diluted with grapefruit oil.