Automated Enzyme Hydrolysis

Greg Stein,

Bristol Myers Squibb

Scientists at Bristol Myers Squibb help develop an automated titration system for PowerPrep Titra-Stat™ applications that provides reliable results with no operator intervention. It offers full data analysis and can be accessed and controlled remotely with a laptop and a phone line.

In the fall of 1995, Bristol Myers Squibb, Syracuse, NY, was interested in replacing several auto-titration systems that technicians were using regularly in the laboratory for pH-Stat enzyme hydrolysis. “The equipment, which was up to fourteen years old, was becoming unreliable,” says Greg Stein, Associate Scientist. “When we contacted the manufacturer to purchase new systems we found they had discontinued our old system and now offered a newer series at roughly twice the price.” Scientists at the company determined that the capabilities of the new system didn’t warrant the investment, so they decided to shop around.

After investigating several systems manufacturers, Stein says, “it became clear that good laboratory auto-titration systems for pH-Stat applications were few and far between.” He eventually opened up a dialogue with Fluid Management Systems, Watertown, MA because “they seemed anxious to understand our application and meet our needs for pH-Stat™ control.” FMS representatives traveled to the Syracuse site three times to design a prototype, from which a new system called the Titra-Stat system and DMS-5500 software were ultimately born. The system provides fully programmable multi-step titrations, real time plotting and automatic documentation of date generated through the entire reaction sequence.

The PowerPrep Titra-Stat™ technique is a well-established procedure that offers real-time analysis and is utilized to regulate and study reactions where hydrogen ions are either consumed or released. These reaction rates can be determined based on the uptake of either an acid or base to maintain a specified pH. Reactions involving metal ions or redox processes can be monitored by changing electrodes. Early PowerPrep Titra-Stat™ systems, such as those used at Bristol Myers Squibb, required the scientist to manually record the titrant volume at predetermined intervals in order to determine the reaction rate. A separate timer was also necessary. Bristol Myers Squibb has been using the Titra-Stat since early 1996, primarily for use in enzyme hydrolysis. Other applications include fixed or stepped pH crystallizations and extractions, and pH control of chemicals baths. Stein says scientists at Bristol Myers Squibb do a great deal of work with Penicillin, so they need a dependable system that can take the drug to the next step in the development process.

The several features that have been found valuable with the PowerPrep Titra-Stat™

1. Control pH of enzymatic reactions that generate and acid. More specifically, an enzyme was capable of splitting a molecule to generate an organic acid. The pH of the environment was maintained within 0.1 units by adding a base solution.

2. The unit was also used to control the pH of a 2 phase acylation where the pH would

have normally been dropping as the reaction if no pH control was used.

3. A staged lowering of pH was used to control crystallizations. For example, an organic acid was crystallized in stages such as holding at pH 5 for 15 minutes. pH 3.5 for 15 minutes and then lowered to pH 2 for the final pH. As these changes were incorporated into the program, the crystallizations could be easily run in a very repeatable manner.

4. The software is easy to operate and helps to develop other methods

The system makes it easy for the user to review data, Stein notes. Titration data, reaction rate, temperature and pump speed curves can all be superimposed on one screen. Saved data from up to four runs can be reviewed simultaneously on the display screen, and can be compressed. The user can display, review and print the results of the run or a compressed summary and analyze reaction trends. Data can be printed in graph and tabular formats stamped with automatic date, time, title, file name and associated titration program. Additionally, the Titra-Stat is the first generation of Remote Monitoring & Control (RMC) systems offered by FMS. This optional package allows the user to monitor and control the system from a remote location using a laptop computer and a standard phone line. The user can monitor pH, temperature, and reaction rates, along with editing the program, reviewing the data files and changing parameters such as pH set points.