FENG LI
CEO
ALLIANCE PHARMACEUTICALS
MALVERN

Years in business: 6

What made you start your business?
I got the idea a long time ago. I worked in same industry as I am now. I got intensive training. I appreciate the experience I got from that company. I was working very hard at learning and thinking about starting the business. By 2006, I began doing research on how to prepare for new business.

What did you do to survive the recession?
Alliance Pharmaceuticals was started in May 2008. We spent almost $1 million, however, only made about $28,000 with three key employees that year. We stuck with our initial business plan, focused on our expertise area, spent time to build up necessary functionalities and prepare for any potential business opportunities. All three employees deferred their pay for a year to ensure the business operation.

What has made your business successful?
We firmly adhere with our business philosophy including professional ethics, scientific excellence, regulatory compliance to win clients trust. We also have maximum business flexibility to understand our clients needs to meet and exceed their expectations.

What is the hardest part of your job?
The regulatory compliance to ensure the data quality and integrity and hiring the right employees.

What advice would you give someone starting their career or looking to start a business?
Have a dream, make a plan, prepare and execute it. You also must be confident enough to be persistent.

Would your advice be any different if you were addressing a minority?
There is no difference. The principle would be the same.

Excerpt from Philadelphia Business Journal

Circulating 25-hydroxyvitamin D3 (25-OHVD3) is widely accepted as the most useful biomarker for evaluating vitamin D status and diagnosing certain diseases. 

Determination of 25-OHVD3 is important during some drug development since the increasing concern of these drugs potentially affecting vitamin D absorption. 

Detecting 25-OHVD3 in biological matrix using LC-MS/MS has been challenged due to its poor ionization efficiency and lack of a predominant daughter ion, which causes a low sensitivity.  Isomeric metabolites of vitamin D and other sterol-like endogenous interferences pose an additional challenge with chromatographic separation.

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

Sample Preparation                                                                  

The samples were prepared in 96-well format by liquid-liquid extraction with MTBE, followed by a 30-minute derivatization with picolinic acid. 25-OHVD3-d6 was employed as internal standard.

LC-MS/MS Analysis

Column: ACE C4, 100 X 4.6 mm, 3 µm particle size

Mobile Phase A: 0.1% formic acid in water

Mobile Phase B: 0.1% formic acid in acetonitrile

Gradient: 0-0.2 min, 75%B; 0.2-3.0min, 75-85%B; 3.05-5.50min, 95%B; 5.55-6.50min, 75%B

Flow rate: 1.5 mL/min

Detector: Sciex API 4000, ESI+

MRM transition: m/z 506.6 à 383.3 for 25-OHVD3

                              m/z 512.4 à 389.3 for 25-OHVD3-d6

Bioanalysis Results

A nitrogen-containing moiety was introduced to the 25-OHVD3 molecule via a derivatization reaction with picolinic acid to increase ionization efficiency (Figure 1).

Derivatization also ensured the formation of predominant product ions that can be used in SRM detections for both 25-OHVD3 and 25-OHVD3-d6 (internal standard) (Figure 2).

Due to high endogenous levels of 25-OHVD3 in blank serum, calibration standards were prepared in a surrogate matrix (5% BSA). Precision and accuracy was evaluated by spiking known concentrations of analyte in pre-quantified “blank” matrix.

Figure 3. Representative chromatograms of 25-OHVD3 and 25-OHVD3-d6 for LLOQ (0.5 ng/mL ) and in surrogate matrix and a rat serum   sample showing endogenous 25-OHVD3.

This method was fully validated with a quantitation limit of 0.5 ng/mL and required only 50 mL of rat serum.  The assay showed excellent linearity (R2>0.998) using a calibration range of 0.5 – 250 ng/mL (Figure 4).

Conclusion 

The derivatization reaction with picolinic acid increased ionization efficiency of the 25-hydroxyvitamin D3 molecule and ensured the formation of predominant product ions, which in turn enhanced the LC-MS/MS sensitivity.

The method was validated as linear, accurate, precise and reproducible. It can be used to determine the concentration of 25-hydroxyvitamin D3 in rat serum as low as 0.5 ng/mL using only 50 mL of sample. 

Scientists

Yinghe Li, Yifan Shi, Meng Fang, and Pam Rogers 

The purpose of this study was to develop and validate a LC-MS/MS method for assaying residual mercaptoethanol in in-process samples of a protein therapeutic product.

2-Mercaptoethanol is a widely used antioxidant in protein production and analysis. As a reducing agent, mercaptoethanol has the ability to cleave disulfide bonds, thus disrupting the tertiary and the quaternary structures of proteins. It is also included in enzyme solution to protect against catalytic site inactivation due to cysteine sulfhydryl oxidation or disulfide formation.

Mercaptoethanol is considered a toxicant, irritating the mucous membranes and respiratory tract, causing sore throat and abdominal pain, and potentially death if severe exposure occurs. As a result, determination of residual mercaptoethanol in biopharmaceutical products becomes crucial for patient safety.

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.

Method

The abundant presence of thiols in protein biopharmaceutical molecules posts the major challenge in analyzing residual mercaptoethanol. We chose LC-MS/MS technique to measure the specific molecule instead of traditional methods which usually only detect the thiol functional group. By derivatizing mercaptoethanol with picolinic acid to form the corresponding dipicolinyl ester (DPE), a detection limit of 4 parts per billion (ppb) was achieved.

The samples were extracted with ethyl acetate, derivatized with picolinic acid, and then extracted with hexane again for analysis. Mercaptoethanol-d4 was used as the internal standard.

LC-MS Conditions

Chromatographic Conditions

HPLC: Shimadzu LC-20AD

Column: Agilent Zorbax SB-C18, 50 x 2.1 mm, 5 µm

Mobile phase A: 0.1% formic acid in water

Mobile phase B: Methanol

Flow rate: 0.6 mL/min

MS/MS Detection

Mass spectrometer: Sciex API 4000

Ionization mode: ESI positive

Source temperature: 500 °C

Ion transition monitored:

Mercaptoethanol-DPE: 289 → 166

Mercaptoethanol-d4-DPE: 293 → 170

Precision and Accuracy of Spiked QCs

Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the limit of detection (4 ppb) in one validation run, and at low (10 ppb), medium (200 ppb) and high concentrations (320 ppb) over three validation runs.

Matrix Effect

Mercaptoethanol was spiked in the matrix as well as in water at n = 6. The matrix effect was calculated as the difference of the mean peak area of the spiked matrix samples and the mean peak area of the pure samples. The method had virtually no matrix effect observed (2.4%).

Derivatization 

The low molecular weight of mercaptoethanol (78 amu) affected selectivity and required careful selection of SRM transitions. The presence of thiol and hydroxyl groups makes it difficult to use reversed-phase chromatography due to the lack of retention. Low organic content during gradient HPLC elution leads to poor ionization efficiency and sensitivity. To address these challenges, mercaptoethanol was derivatized with picolinic acid to form a dipicolinyl ester, which a limit of detection at 4 ppb was successfully achieved.

Conclusion

• A novel LC-MS/MS method capable of assaying residue mercaptoethanol in in-process samples of a protein therapeutic product was developed and validated.
• Matrix interferences or unknown impurity peaks are readily removed by two steps of liquid-liquid extraction.
• Derivatization with picolinic acid increases the assay sensitivity to reach a detection limit of 4 ppb.
• The method was demonstrated to be accurate, specific for 2-mercaptoethanol and reproducible.

Scientists

Yifan Shi, Yinghe Li, Meng Fang, William F. Wagner, Aston Liu, Sandro X. Nalli