The extent of binding to proteins is an important parameter to assess when screening drug candidates during in-vitro ADME studies. Assessing the free fraction of drug available in the biological system is critical to understanding its distribution potential, as it is generally believed that the free fraction drives therapeutic outcomes since it is the free drug (that which is not bound to proteins) that can move to and interact with the sites of action, and high binding to proteins can affect the metabolism and elimination of the drug.

          There are three methods most commonly used to assess protein binding, and it is important to assess their advantages and disadvantages when selecting methods to use for screening studies.

Rapid Equilibrium Dialysis (RED) makes use of a device with two chambers separated by a semipermeable membrane. Spiked matrix is added and over an incubation period, the free drug comes to equilibrium across the membrane while proteins and protein-bound drug cannot move across the membrane; free fraction is calculated by comparing the concentration of drug from the protein and protein-bound drug side to the protein-free, free drug side.

Advantages: Most common method (easy cross-comparisons), simple apparatus/setup and high throughput

Disadvantages: Requires long-term stability in the matrix (typical incubation period is 4 hours), nonspecific binding to the device/membrane, volume shifts and protein leakage over the incubation period.

Ultracentrifugation (UC) uses high-speed centrifugation to precipitate proteins and protein-bound drug from a spiked matrix; free fraction is calculated by comparing the concentration of drug in the protein-free supernatant and the intact spiked matrix.Advantages: Avoids membrane-related issues (nonspecific binding, leakage across membrane, etc.)

Disadvantages: Usually low-throughput, harder to control pH, temperature (conditions that can significantly affect protein binding), requires longer-term stability in matrix (typical centrifugation time is two hours), the supernatant may not be completely protein-free, leading to an artificially high free fraction observed.

Ultrafiltration (UF) uses a two-chambered device separated by a semipermeable filter. Spiked matrix is loaded into the device and centrifuged at moderate speed, during which time free drug passes through the filter while proteins and protein-bound drug cannot; free fraction is calculated by comparing the concentration of drug in the protein-free filtrate and the intact spiked matrix.

Advantages: Suitable for unstable compounds (typical centrifugation time is 30 minutes), quick and technically simple, high throughput/useful to screen many compounds to rank free fraction

Disadvantages: Nonspecific binding to the filter membrane or collection chamber (under-estimation of free fraction), molecular sieving may occur (liquid moves through filter more quickly than the drug, under-estimating free fraction)

          An enzyme-linked immunosorbent assay (ELISA) is a chemical test to detect the presence of proteins or other molecules. The enzyme-labeled antibody binds to antigens leading to a color change that is used to quantify antigen-antibody interactions. Quantification of these concentrations requires measuring the intensity of the color change, which indicates the concentration of antigen bound to the antibody. ELISA’s include four variants: direct, indirect, sandwich, and competitive, which all have their respective advantages and disadvantages.

Benefits of an ELISA

High Sensitivity: The concentration of both antibody and antigen can be quantified on the nanogram scale. This means that scientists can use a smaller amount of enzyme, which still produces a large scale catalytic reaction.

High Specificity: Antibodies are very specific to their targets due to their spatial configuration and chemical structure. They can easily recognize and bind to antigens similar to how a key unlocks a specific door.

Versatility: Indirect ELISA’s allow for multiple primary antibodies created in one species with the same labeled secondary antibody used for detection. Also, it’s easy to wash off any unbound antibodies by applying a detergent solution to the plate.

Clinically approved ELISA tests for diseases like HIV, Lyme disease, and tuberculosis are proving their efficacy and value to the Pharmaceutical industry.

AAPS Poster - #M1430-03-017 - Development of an LC-MS/MS Method for Quantification of Cytarabine in Rat Plasma - ePoster presentation track: Bioanalytics - Forum3 - Screen 17 - Date/Time 2018-11-05 2:30

Alliance Pharma is gearing up for the #AAPS PharmSci 360 Conference in DC.  Meet Our Scientist for Poster  #M1430-03-017 - Development of an LC-MS/MS

Method for Quantification of Cytarabine in Rat Plasma. Stop by our booth #610 or schedule a meeting with us at the conference Ryan Klein 919-801-3146 or email rklein@alliancepharmaco.com.

It’s a great opportunity to connect with leading #pharmaceutical #scientists from across the globe!

Alliance Pharma is dedicated to bringing comprehensive solutions to our collaborative partnerships.  We understand the importance of efficiency and our team of scientists strive to provide the highest analytical services for your project. 

Our recent purchase of multiple Agilent 6545XT AdvanceBio QTOFs – which is specifically designed with biopharmaceutical characterization in mind - provides high resolution and sensitivity for qualitative and quantitative analyses of biomolecules ranging from peptides to intact monoclonal antibodies. The iterative peptide mapping workflow allows us to dig deep into samples with a wide range of analyte concentrations (i.e. host cell proteins and sequence variants). The Agilent 6545XT AdvanceBio QTOF maximizes our uptime by performing thousands of protein injections without degradation of performance with mass accuracies within 10 ppm for glycosylated intact proteins. 

At Alliance Pharma we believe keeping up with the latest technology helps us to provide the best services for our clients.  The 1290 Infinity II LC system when coupled with the 6545XT, represents the next generation LC for ultrahigh-performance liquid chromatography with superior reliability, offering efficient and reproducible separations of biomolecules for detection by MS or integrated UV and FLD modules. 

By using instruments that complement our scientist’s knowledge and experience we meet analytical objectives, increase productivity, and are cost-effective.  Contact us to see how our recent instrument purchase will help keep your project on track.  Call or email Ryan at 919-801-3146 or  rklein@alliancepharmaco.com for more information.

Alliance Pharma has hired Dr. Ryan Klein as Director of Business Development. Dr. Klein has more than 20 years of experience in the pharmaceutical industry developing both oral and topical dosage forms for a range of indications. Dr. Klein began his career at GlaxoSmithKline, where he was an integral component of their drug discovery organization providing drug metabolism and pharmacokinetic expertise to project teams. He was instrumental in the development and implementation of a number of in vitro and in situ ADME models to assess drug absorption, metabolism, and disposition.

After leaving GSK, Dr. Klein joined Tergus Pharma, a CRO providing topical R&D services to the pharmaceutical industry. At Tergus, Ryan filled various roles including leadership positions as the Head of Research & Development and Head of In Vitro Sciences, as the company grew from five employees to approximately seventy five over a seven year period.

Dr. Klein’s areas of expertise include drug permeability, absorption and metabolism in the gastrointestinal tract, liver and skin, intestinal drug transporters, and the anatomy, physiology, and pharmacology of the skin and GI tract. He also has a strong analytical background with extensive experience developing and validating in vitro release testing methods for semi-solid dosage forms, as well as developing and validating HPLC assay and impurities methods.

Leveraging Dr. Klein’s experience on the client side allows Alliance Pharma to collaborate on a higher level with Sponsors, using his in-depth knowledge and vast experience to align the best team of scientists to support each project.

Ryan earned his bachelor’s degree in Chemistry from Wake Forest University in Winston-Salem, North Carolina, and earned his doctoral degree in Pharmaceutical Sciences from the School of Pharmacy at the University of North Carolina at Chapel Hill. Ryan has authored & co-authored numerous scientific publications and patents, and serves as a member of several pharmaceutical research focus and discussion groups.

To read more or to connect with Ryan, view his LinkedIn profile https://www.linkedin.com/in/ryanrklein/

Colin Barry, Ph.D. & Scott Ugrin, Ph.D.

Alliance Pharma’s bioanalytical team provides comprehensive services for both small and large molecules using a variety of platforms from mass spectrometry to ligand binding assays using an assortment of specialized instrumentation. Our analytical laboratories boast thirteen LC-MS/MS systems from three different manufacturers and include various platforms. Nine systems from AB Sciex are each coupled to Shimadzu UHPLC systems, including two API4000 QTRAP systems, two API4000 triple quad systems, three 5500 triple quad systems, and two 6500 triple quad systems. Each system is designed for high sensitivity quantitative analysis of small molecules generated from either in vitro or in vivo studies and in various matrices.

Alliance Pharma’s small molecule mass-spec based bioanalytical team specializes in:

• Development, optimization, and transfer of bioanalytical assays
• Method Validation
• Sample analysis to support clinical (GLP) and non-clinical studies
• Plasma, serum, whole blood, and all tissue types
• Biomarker assay development, validation and sample analysis
• High throughput screening from in vitro ADME assays
• Metabolite identification

The newest additions to the bioanalytical lab include two Agilent 6545 XT Q-ToF high resolution mass spectrometers coupled to Agilent 1290 Infinity II UHPLC systems with PDA and FL detectors. These new systems add additional capacity to our existing high-resolution accurate-mass (HRAM) systems: 1) a Dionex RSLCnano, 2-D nano/micro/cap UHPLC system coupled to a Q Exactive Plus, and 2) an Orbitrap XL from Thermo Scientific. The newest HRAM systems are ideally suited for analysis of proteins and peptides and will allow generation of highly detailed information for intact proteins and antibodies, confirmation of sequence at the peptide level, and sophisticated analysis of both simple and comprehensive post-translational modifications.

The combination of mass accuracy (> 1 ppm) and high resolution (up to 280,000 at m/z 200) allow for increased specificity and sensitivity in extremely complex matrices. The additional capabilities position Alliance Pharma to continue to expand our large molecule qualitative and quantitative analysis capabilities.

Chunying Gao, Ph.D.

Drug transporters play a key role in the absorption, distribution, metabolism, elimination and toxicity (ADME-Tox) of many drugs by controlling the entry or exit of drug molecules into and out of tissues or organs. Due to the importance of drug transporters in these processes and the potential drug-drug interactions among drug molecules on these transporters, in vitro transport studies on a standard panel of drug transporters are recommended by the FDA, EMA, and PDMA regulatory agencies to evaluate the potential interactions as substrates and/or inhibitors among the investigational drugs. The current panel includes P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1 and MATE2-K; although regulatory agencies are routinely requesting data on additional transporters outside of the current group. 

According to the FDA guidance,^[1] several in vitro models are recommended for each transporter:

Alliance Pharma has established a set of cell- or membrane vesicle-based assays to support filing packages for the studies recommended by regulatory agencies. For ABC transporters, a membrane vesicle assay (for P-gp and BCRP) and MDR1/MDCK bidirectional permeability assay (for P-gp) has been established. For SLC transporters, an assay platform based on transiently transfected HEK293 cells has been optimized and validated, and can be used for both screening of substrate/inhibitor and K_m/IC₅₀ determination.

[1] https://www.fda.gov/downloads/Drugs/Guidances/UCM581965.pdf

This method was validated by following the US FDA guidance for bioanalytical method validation. To read more about the Validation of an LC-MS/MS method for simultaneous quantification of venlafaxine and its five metabolites in rat plasma and its application in a pharmacokinetic study click here.

Guodong Gua,⁎, Michelle Blacka, Colt Cooksona, Anna Fiorellaa, Yinghe Lib, Steven H. Gormanc, Ray Bakhtiarc

a Alliance Pharma, Inc., Malvern, PA, United States

b GlaxoSmilthKline (GSK), Collegeville, PA, United States

c Teva Branded Pharmaceutical Products R&D, West Chester, PA, United States

Journal of Chromatography B 1087–1088 (2018) 29–35

Scientists who specialize in developing mass spectrometric methods know that the methods must be individually tailored to successfully support a pivotal clinical trial or to provide bioanalytical support along any phase of the drug development process. A specific mode of mass spectrometry called multiple reaction monitoring (MRM) is a powerful tool because it uses multiple mass spectrometers in tandem to increase sensitivity and selectivity of analysis, generating the most reliable data. Read on to find out why this matters to experts in the fields of pharmacokinetics, bioequivalence, toxicity, and bioanalysis.

Extreme Sensitivity

Mass spectrometry has the ability to accurately determine concentrations of biomarkers present in human matrices down to nanomolar levels. D-erythro-Sphingosine-1-phosphate (S1P) is a biomarker for certain diseases such as atherosclerosis and cancer.

Method Development and Validation for the Quantification of D-erythro-Sphingosine-1-phosphate (S1P) in Human Plasma Using LC-MS/MS

Versatility and Customization

Instrumentation techniques are customizable. Liquid chromatography tandem mass spectrometry has the ability to monitor several different classes of compounds. There are intricate parameters of mass spectrometry that can be optimized for every study. See below how LC-MS/MS techniques have been used to analyze compounds like cholesterol and ManNAc, which are endogenously present in plasma, and goserelin, a synthetic hormone used to treat prostate and breast cancer.

Quantification of 4β-Hydroxycholesterol and Cholesterol in Human Plasma Using LC-MS/MS

Method Development and Validation for the Quantitation of ManNAc in Human Plasma Using HILIC LC‑MS/MS

A Rapid and Sensitive Method for the Quantification of Goserelin in Human Plasma Using HPLC-MS/MS

Speed

By coupling liquid chromatography with mass spectrometry, concentrations of several compounds in a sample can be achieved in a run as short as 3 minutes. Whether you are interested in analyzing several different drugs or a parent compound and its metabolites, it can be done in the same run making study conduct very efficient.

SHORTCUT to CYP Enzyme Activity Monitoring

In March, thirty-two Alliance Pharma employees competed in the First Annual Ping Pong Madness Championship. The games commenced at a neck-breaking pace of back and forth and culminated with a title upset that only two bracket participants predicted. Fierce competition, a pizza party, and a pep rally made the experience a WIN for all employees, even though only one person took home the title of PING PONG MADNESS CHAMPION. 

Congratulations Yong! 2017 Ping Pong Madness Champion

The skills, that stance! It's no wonder YONG went all the way to the top of the Ping Pong Madness Bracket! https://t.co/83di6ThG94

— Alliance Pharma (@Alliance_Phar) March 24, 2017

Here's a two minute clip from the match. Watch Yong's killer spike as early as 9 seconds into the video! Dr. Lin also attributes his excellent pipetting skills to his ping pong talent.  

Our spirit team really pumped up the crowd. Here's a video of the cheer that brought everyone to... cheers:

 You can listen to the Ping Pong Madness playlist on Spotify.