As we approach graduation season, many college seniors are beginning their bioanalytical job search on campus, online, and by networking. Entry-level jobs offered at Alliance Pharma are suitable for students with a Bachelor's or higher in the sciences such as Chemistry, Biochemistry, or Biology. New scientists thrive in this environment, where they have direct access to Ph.D. level mentors in their day-to-day lab operations. Our company also has several career paths with two stages for Analysts and two stages for Scientists and opportunities for advancement. 

Reasons to apply for bioanalytical jobs at Alliance Pharma:

• Rapid Growth - Dr. Feng Li, President, is a two-time winner of the Smart CEO Future50 award
• Career Support - Working in a small pharmaceutical company setting gives novice researchers access to more complex and challenging assignments.
• Mentorship - Alliance Pharma prides itself on the ability to hire young talent and nurture them through the initial stages of lab work. 

In the video below, Kale Drost (Ursinus '15) and Anna Fiorella (Ursinus '16) share their post-college career experience. They offer anecdotes of working in the bioanalytical laboratory and demonstrate a few scientific study processes. Be sure to listen as to what you can expect in the real world in the words of two Alliance Pharma employees. 

“Enjoy the time you have left in school because you will miss it.” -Anna

Here are 5 tips to make a GREAT Impression with your job resume

1. Check for typos or misspellings.
2. Include any relevant volunteer work and honors.
3. Use the keywords scientific researchers seek in new hires- LC-MS/MS, mass spectrometry, sample extraction, instrument maintenance, etc.
4. Don't over-exaggerate your lab experience.
5. Keep it to one page, especially if you are an entry-level candidate with a Bachelor's degree.

Get to know Alliance by following our LinkedIn page for career postings and frequent updates. We also post job openings directly on our site. 

Over the years, our scientists have been tasked with a variety of complex LC-MS/MS studies. In their research, they worked to produce cost-saving procedures, deliver reproducible results, and employ automation technologies. 

Here is an overview of the Bioanalytical study research presented at scientific conferences over the past four years. Click the links to view the full poster; including analysis, details, and conclusions.

1.) Prove Microsampling accuracy with LC-MS/MS 

Meng Fang shares our microsampling study. Download the poster from the app or stop by booth 1255. #aaps2016 pic.twitter.com/p5jwzmBCxI

— Alliance Pharma (@Alliance_Phar) November 15, 2016

Mouse Pharmacokinetic Study of Ceftriaxone Using Mitra™ Microsampling Devices and LC-MS/MS

In this study, an experiment was designed to compare a serial blood sampling method using Mitra™ microsamplers with parallel blood sampling methods using both Mitra™ microsamplers and venipuncture.

2.) Automate the work of quantifying S1P in Human Plasma using LC-MS/MS

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

An automation friendly LC-MS/MS method with a calibration range of 10 to 400 ng/mL was developed and validated to support clinical trials using S1P as a biomarker.

3.) Quantify HMF and HMFA in Human Plasma with LC-MS/MS

Method Development and Validation for the Quantitation of HMF and HMFA in Human Plasma Using LC-MS/MS

A method for the quantitation of HMF and one of its major metabolites, 5-hydroxymethyl-2-furoic acid (HMFA), has been developed and validated in commercially available human plasma by Alliance Pharma. 

4.) Confirm stability with rapid and sensitive LC-MS/MS method

SHORTCUT to CYP Enzyme Activity Monitoring

A rapid and sensitive LC-MS/MS method was validated for cortisol and 6ß-HC analysis
in human urine. Method accuracy, precision, repeatability, selectivity, F/T stability, processed sample stability, bench-top stability, and long term stability have been confirmed.

5.) Evaluate drug-drug interactions during LC-MS/MS clinical studies

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

The plasma concentration ratio of 4b-hydroxycholesterol (4b-HC) to cholesterol has been recognized as a reliable marker for the assessment of Cytochrome P450 (CYP) 3A4 activity. It could be a valuable yet simple and cost effective side-product of a clinical study to evaluate CYP3A4-mediated drug-drug interactions.

6.) Support a Phase I clinical study with an LC-MS/MS method

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

The purpose of this study was to develop and validate an LC-MS/MS method for assaying N-acetylmannosamine (ManNAc) in human plasma (K2EDTA) to support a phase I clinical study.

7.) Measure Goserelin in human plasma using LC-MS/MS

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

The purpose of this study was to develop and validate a rapid and sensitive LC-MS/MS method for measuring Goserelin in human plasma (K2EDTA).

8.) Determine the quantification of 25-hydroxyvitamin D3 with an LC-MS/MS method

Quantification of 25-hydroxyvitamin D3 in Rat Serum Using Derivatization to Enhance LC-MS/MS Sensitivity

In this study, a sensitive and robust LC-MS/MS method was developed and validated for the determination of 25-OHVD3 in rat serum.

9.) Quantify Mercaptoethanol through LC-MS/MS analysis

Quantification of 2-Mercaptoethanol in Bulk Drug Substance by LC-MS/MS

Here, we report a LC-MS/MS method that utilizes derivatization with picolinic acid to quantitatively analyze residual mercaptoethanol and has been successfully used in biopharmaceutical manufacture.

For a second year in a row, Alliance Pharma was honored as one of Philadelphia Region’s Future 50 award winners by SmartCEO magazine.  The program recognizes the region’s fastest growing, midsized companies based on a 3-year average of employee and revenue growth.  This year’s Future 50 winners collectively generate nearly $4.84 billion in annual revenue and employ 25,272 individuals in the Greater Philadelphia area.  Frank Li, PhD, President of Alliance Pharma, attributes his repeat success with putting clients first, understanding their goals, and delivering the service they expect.

Pictured: Frank Li (center), with Jeannette Bezinque and Tom Kohl. Photo by: David Michael Howarth Photography

"We put our clients first and strive to build a trusted partnership by offering top-quality service, competitive pricing, and quick turnaround time. We also strive to maintain the highest standards of professional ethics, scientific excellence, and regulatory compliance." - Frank Li, President, Alliance Pharma

In the magazine, Frank goes on to describe the importance of being agile and adaptive to the shifting needs of clients.  He also strives to empower all employees to reach their full potential.

Watch Frank Li accept a 2017 @smartceo #Future50 Award from SmartCEO Magazine. The winners from the Philadelphia... https://t.co/bdkGi3bG0e

— Alliance Pharma (@Alliance_Phar) January 18, 2017

Read the full interview here: http://www.smartceo.com/2017-philadelphia-future-50-alliance-pharma-inc/

See Frank in the Group 1 video starting at 2:19 here: https://www.youtube.com/watch?v=3kjHOgWUfNc

In 2016, Alliance Pharma had many notable achievements. One of the reasons that we continue to see growth year after year is the strong leadership of Dr. Feng (Frank) Li. For a second year in a row, Alliance Pharma was recognized with SmartCEO Future 50 award as Philadelphia region’s fastest growing mid-sized companies for 2017. From an increase in laboratory space and personnel to a host of new instruments, Alliance Pharma’s expansion facilitates an agile and adaptive environment to meet the specialty research needs of our clients—some of the world's best pharmaceutical companies.

By opening the laboratory animal facility in April 2016, Alliance began to offer in-life dosing studies and created three vivarium technician jobs. We also added critical research space for light-sensitive compounds. Nearby, a new temperature and humidity control unit upgraded our archive room storage capabilities. A few months ago, a crane arrived to deliver an AC unit for the new large-molecule mass spectrometry laboratory. In addition to space enhancements, we also added several new pieces of equipment.

Brand New Equipment Purchased in 2016

• Thermo Scientific Q Exactive Plus High-Resolution Mass Spectrometer
• Another Hamilton Robotics Automated Liquid Handling System
• Another Meso Sector Imager S600 Microplate Reader  
• Three additional High-Performance Liquid Chromatography (HPLC) systems
• Five additional laboratory freezers
• Beckman Coulter AU480 Chemistry Analyzer

System Formalization

As part of our commitment to continuous improvement in service, we updated our quality manual and disaster recovery plan. 

A new software system was implemented to facilitate project management for each department. We also implemented a secure, digital sample management system to easily track data about storage and shipments. Laboratory operations has made improvements in the instrument inventory system, chemical and solvent inventory system, reagent inventory system, and HPLC column tracking system. Also, we have partnered with excellent providers to streamline our accounting and human resources systems.

Finally, a Training Coordinator was appointed to improve our on-boarding process for new employees and to identify areas for staff training and development. Since our company grew by over 50%, this was a much-needed organizational structure.

New Hire Spotlights

Our workforce grew to a total employee count of 63 in 2016. You can check out some of our staff announcements on our LinkedIn page. Our leadership team additions this year include Dr. Weiqing Chen, Dr. Wei Lu, and Dr. Colin Barry.

Notable Achievements in Social Media

On December 29th, we relaunched our site with Hubspot to deliver innovative and helpful content about the pharmaceutical research industry.

We gained our 100th Twitter follower on December 15. Thank you @ArcturusRX!

Eight team members worked together to create our first social media video, the #mannequinchallenge.

Over 1000 people saw our October 19 LinkedIn update.

Check us out on LinkedIn, Twitter, Facebook, or on our blog to read more about this year’s highlights. 

Future Direction

Our promise for 2017 is to continue to strengthen our specialties. We will continue to emphasize quality, compliance, and skill training. It is important for us to continue to improve our operational efficiency.  We plan to achieve new levels of productivity by further standardizing and harmonizing processes, and automating procedures, wherever possible. As always, we seek to deliver the highest quality data and in the most timely expectations. 

In 2016, Alliance Pharma conducted a Customer Survey to measure satisfaction of our bioanalytical, biopharmaceutical, and drug metabolism services to our clients. Our goal was to assess how we were performing in relation to our business philosophy. Since we measure our success based on the success of our clients, it is important that we understand the areas of our business that are working and the areas that need improvement.

Overall, 100% of clients who responded to our survey gave Alliance Pharma a rating of SATISFIED or higher in regards to questions about services, personnel, performance, support, management, and costs. In fact, 75% of clients stated that they are VERY SATISFIED with Alliance Pharma.

The Customer Survey went out to Scientific Directors, Senior Research Directors, Assistant Scientists, Senior Managers, and Professors. When these clients were asked, “How satisfied are you with the performance of our technicians?” 70% replied that they are VERY SATISFIED. In addition, 65% of our clients responded that they are VERY SATISFIED with our management’s commitment to assist.

Customer Survey Data

We also used the survey to understand why our clients are choosing to partner with us. We know that there are many CROs in the marketplace, so we value each and every client. The replies showed that our network is strong and that referrals are the number one factor for our clients choosing to partner with us. The survey also revealed that our clients are choosing us for other reasons, as follows:

• Timing
• Cost
• Quality
• Location
• Assay Capability

Customer Focused Improvements

Based on the results of the survey, we plan to update our website design and functionality. We will also create new company literature to describe the full extent of our services, especially those that we have added this year, such as the Laboratory Animal Facility and Large Molecule Bioanalysis.

Alliance Pharma would like to thank all of our customers for their time in completing the survey to help us understand how we can better serve all your needs! We are committed to delivering the highest quality services at a competitive price, and thus build a lasting partnership. Thank you for your continued patronage.

On November 13, our team of top scientists traveled to Denver, Colorado for the 2016 AAPS Annual Conference for pharmaceutical scientists. The conference was kicked off with a keynote address from Daniel Fletcher, PhD who discussed novel technology, new delivery modalities, accelerated drug development, and precision medicine. Our booth, which was serendipitously situated next to the Rest and Relaxation Lounge, welcomed a host of pharmaceutical professionals, academic leaders, and job-seekers. Visitors received an Alliance Pharma lunchbox or a handy letter opener.

Many connections were made at the various networking events like the Welcome-Reception, the American Chinese Pharmaceutical Association Meeting, and the 80’s Party hosted by Core RX and Mutchler Inc. Presenters and attendees at the conference included Teva, BASF, Agilent, Triangle Research Labs, Science Exchange, MedPace, and Pfizer, to name a few. Many members of the FDA were also onsite.

Researchers seeking a CRO (contract research organization) find value in the conference because they can make a trusted in-person connection and quickly discern the qualities of a specific CRO. For example, the ideal CRO offers quick-turn around, high-quality data, and competitive pricing. The conference was also an opportunity to share updates on our company. This year, Alliance Pharma had added a Vivarium for animal research studies and a new lab for Large Molecule Bioanalysis.

Tweets from #AAPS2016

Dr. Meng Feng presented a poster on microsampling titled Mouse Pharmacokinetic Study of Ceftriaxone Using Mitra^TM Microsampling Devices. (View the full details here.) In his work with a group of Alliance colleagues, Dr. Feng concluded that Mitra^TM microsampling devices provide a methodology that can be used for serial blood sampling in drug discovery mouse studies, and which could reduce costs, improve animal welfare, and save precious test articles.

Dr. Yong Lin presented a poster on Adaptation and Validation of a Multiplex Assay Kit for the Quantitative Analysis of Aβ38, Aβ40, and Aβ42 Peptides in Cynomolgus Monkey Plasma at AAPS. (View the original poster here.)

2017 Conference for Pharmaceutical Scientists

If you are seeking another conference for pharmaceutical scientists, consider joining us in 2017 at one of our upcoming events. View the full conference schedule here.

Purpose

Blood sampling is a common and important specimen collection procedure used in research. One of the most commonly used animals in drug discovery pharmacokinetic (PK) studies are mice. Animal research guidelines exist that limit the frequency and amount of blood that can be collected from a single animal. In mice, due to their limited body size, parallel blood sampling is generally used whereby multiple mice are subjected to a single blood draw through cardiac puncture. Oneissue with parallel sampling, however, is that the number of mice required for a study can be large. Consequently, the amount of test compound required for doseadministration will also be large.
Repeated or serial sampling in a single animal, on the other hand, is often difficult, especially when cannulation is not an option. Mitra^TM microsampling devices (Neoteryx LLC) offer an alternative method for serial blood sampling in mouse PK studies. Using serial blood sampling rather than parallel blood sampling may greatly reduce the number of animals needed and lead to more reliable data by excluding individual differences. In this study, an experiment was designed to compare a serial blood sampling method using Mitra microsamplers with parallel blood sampling methods using both Mitra microsamplers and venipuncture.

Method

Animal Experiment Design

Species: CD-1 mice
Compound: Ceftriaxone
Dosing: 1 mg/kg IV, single dose (Groups 1 and 2);
5 mg/kg PO, single dose (Groups 3 and 4)
Vehicle: 0.9% sodium chloride (saline) solution
IV Time Points: 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 hours
PO Time Points:0.25, 0.5, 1, 2, 4, 7, and 24 hours
•Group 1 (IV) and Group 3 (PO)-Parallel Sampling (3 mice per time point per dose route) - At each time point, blood samples (10 μL) were collected from the lateral tail vein of each mouse using Mitramicrosamplers, and blood samples were collected by single venipuncture for plasma analysis.
•Group 2 (IV) and Group 4 (PO)- Serial Sampling (3 mice per dose route) - At each time point, blood samples (10 μL) were collected from the same mice using Mitra microsamplers

Mitra Sample Extraction

•Mitra microsampler tips were removed and soaked in 100% water to achieve better recovery of relatively polar compound ceftriaxone.
•Protein was precipitated using acetonitrile with internal standard (d3-ceftriaxone). Supernatant was dried down and reconstituted before injection.

LC-MS/MS Conditions

UHPLC : Shimadzu Nexera X2 LC-30AC
Column: Agilent Zorbax SB-C18
Mobile Phases: A: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile
Mass Spectrometer: AB SCIEX Triple Quad TM5500
Ion Transitions: m/z555.1→396.1for ceftriaxone
m/z558.1→399.0 for d3-ceftriaxone

PK Calculations

Noncompartmental models using the Phoenix®WinNonlin®software

Conclusion

• Extraction method from Mitra microsampling devices was optimized to improve the recovery of ceftriaxone, a relatively polar compound.
• Comparable concentration results and PK parameters were obtained using the Mitra microsampling method and traditional blood sampling method after both IV and PO dosing.
• Mitra microsampling devices provide a viable alternative for serial blood sampling in mouse drug discovery studies.
• The use of Mitra microsampling devices could reduce costs, improve animal welfare, and save precious test articles.

Acknowledgements

Meng Fang, Gordon Gu, Brandon Milan,Bobby Virasingh, Ashley Groff, Jamie Freed, Catherine Clifton, Deping Cheng, Yinghe Li
Alliance Pharma, Inc., 17 Lee Boulevard, Malvern, PA 19355
Neoteryx LLC, 421 AmapolaAvenue, Torrance, CA 90501

D-erythro-Sphingosine-1-phosphate(S1P), a bioactive lysophospholipid, is an important mediator of inflammation, atherosclerosis, and cancer. S1P is proposed as a clinical biomarker and diagnostic variable in fundamental research, routine testing, and large-scale clinical trials due to its signaling transducer functions.

An automation friendly LC-MS/MS method with a calibration range of 10 to 400 ng/mL was developed and validated to support clinical trials using S1P as a biomarker.

Sample Extraction

Challenge in Quantitative Recovery

The bipolar structure of S1P presented a challenge during extraction due to its tendency to accumulate in the aqueous/organic interface.

Overall recovery was 91.3% for S1P and 109.5% for S1P-d7.

Extraction Method

• A 50-uL sample size was used with internal standard S1P-d7. 
• Recovery was improved by using a liquid-liquid extraction with 10% formic acid (FA) in methyl tert-butyl ether (MTBE) as a solvent. 
• Partial supernatant was dried, reconstituted, and injected into the LC-MS/MS system.

LC-MS/MS Method

LC-MS/MS Conditions

HPLC: Shimadzu LC-20AD

Column: Thermo Acclaim C8, 50x2.1mm, 5um

Mobile phase A: 0.1% formic acid in water

Mobile phase B: 0.1% formic acid in acetonitrile

Flow rate: 0.8 mL/min

MS/MS Detection

Mass Spectrometer: AB Sciex API 4000

Ionization mode: ESI positive ion mode

Source temperature: 500° C

Ion transition monitered:

S1P: 381 → 264

S1P-d7: 388 → 271

Results

S1P Calibration Curve

The assay showed a linear calibration range of 10 to 400 ng/mL. The curve was linear (R² > 0.998) using weighted 1/x² regression. 

Chromatography and Method Sensitivity

Representative chromatograms of the quality control at the lower limit of quantification (LLOQ-QC, 10 ng/mL) and blank surrogate matrix (2% bovine serum albumin [BSA] cleaned with active charcoal).

Matrix Effect

No matrix effect was observed when QCs prepared in matrix were compared with those prepared in neat solution

S1P Endogenous Concentration

The endogenous level of S1P in 6 lots of screened blank matrix ranged from 19.1 to 157 ng/mL. A single lot was quantified (150 ng/mL) and used to prepare MQC (endogenous level) and HQC (200 ng/mL + endogenous level) samples.

Conclusions

• A sensitive, selective, and automation friendly method capable of assaying S1P in human plasma was developed and validated to support clinical trials using S1P as a biomarker.
• Excellent recovery of S1P was achieved by adjusting the extraction solvent composition.
• The method could be applied to other phospholipids that pose similar challenges in quantitative recovery.

Acknowledgement

Guodong (Gordon) Gu, Michelle Black, Deping Cheng, Yinghe Li, Yifan Shi, Meng Fang, Lynn Maines
Alliance Pharma, Malvern, PA; Janssen Research and Development, Spring House, PA; Apogee Biotechnology Corporation, Hummelstown, PA

This study was financially supported by Apogee Biotechnology Corporation.

5-Hydroxymethylfurfural (HMF) is a water-soluble heterocyclic organic compound derived from sugars. HMF binds with high affinity to intracellular sickle hemoglobin (HbS). In vivo studies using transgenic sickle mice showed that orally administered HMF inhibits the formation of sickled cells in the blood. NIH and its collaborators conducted investigations into the possibility of HMF as a treatment of Sickle cell disease (SCD) which is characterized by an abnormality in the oxygen-carrying haemoglobin molecule in red blood cells. A method for the quantitation of HMF and one of its major metabolites, 5-hydroxymethyl-2-furoic acid (HMFA), has been developed and validated in commercially available human plasma by Alliance Pharma.

Method

The standards and QCs were spiked with stable-isotope labeled HMF/HMFA as internal standards and extracted by protein precipitation with 0.1% formic acid in acetonitrile in a phospholipid removal plate. The eluent was evaporated to dryness and the residue was reconstituted with acetonitrile:water (10:90). The analysis was conducted utilizing a Schimadzu Prominence 20AC HPLC system coupled with SRM detection in ESI positive mode for HMF and in ESI negative mode for HMFA on a Sciex API 4000 Q Trap mass spectrometer. Chromatographic separation was achieved using an Atlantis T3 column with 0.02% acetic acid in water and 4 mM ammonium formate in methanol as the mobile phases.

LC-MS Conditions

Chromatographic Conditions
HPLC: Shimadzu LC-20AC
Column:Waters, Atlantis T3, 100x2.1mm, 3 μm
Column Temperature: 40oC
Mobile phase A: 0.02% acetic acid in water
Mobile phase B: 4 mM ammonium Formate in methanol
Flow rate: 0.6 mL/min

MS/MS Detection

Mass spectrometer: Sciex API 4000 Q Trap
Source temperature: 550oC
Ion transition monitored:
HMF: ESI positive mode
m/z 126.9 → 53.1
HMFA: ESI negative mode
m/z 141.0 → 69.2

Precision and Accuracy of Spiked QCs for HMF

Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the lower limit of quantification (5 ng/mL) in one validation run, and at low (15 ng/mL), medium (150 ng/mL) and high concentrations (1500 ng/mL) over three validation runs.

Precision and Accuracy of Spiked QCs for HMFA

Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the lower limit of quantification (0.1 μg/mL) in one validation run, and at low (0.3 μg/mL), medium (6 μg/mL) and high concentrations (75 μg/mL) over three validation runs.

Conclusion

• A selective and sensitive HPLC-MS/MS method for the quantification of HMF and HMFA in commercially available human plasma was developed. 
• Great retention and selection of highly hydrophilic compounds were achieved using carefully selected HPLC column and optimized mobile phases.
• Phospholipid removal plate was used to decrease the ion suppression resulted from the phospholipids in the protein precipitation extract.
• The method was validated as linear, accurate, precise and reproducible.

Acknowledgements

Meng Fang, Yifan Shi, Yinghe Li, Michael Zhang, Bradley Gillespie, Warren Stern, Amy Wang, Nora Yang, and Xin Xu
Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355; Leidos Biomedical Research Inc., Frederick, MD 21701; AesRx, LLC, Newton, MA 02466; TRND, National Center for Advancing Translational Sciences, NIH, 9800 Medical Center Dr., Rockville, MD 20850

On Sunday, May 3, 2015, Alliance Pharma participated along with 40,000 people in Boston’s 47th annual “Walk for Hunger.” The 20-mile route wound around Boston and surrounding communities and raised money to create sustainable solutions for hunger in Massachusetts. This year’s walk raised more than $3 million. Alliance Pharma raised $500 and walked the whole 20 miles to support this great cause. Thank you for your donations and support!