The complex manufacturing processes that are inherent in biotechnology and pharmaceutical manufacturing require advanced instrumentation to ensure an optimal path to the final product. Mitigating risk throughout these processes is the key to increasing profits. The Prima PRO Process Mass Spectrometer offers the speed and precision necessary to reliably track process dynamics, enabling timely corrective action to be taken. From research and development to creation of the final product, the Prima PRO process mass spectrometer helps bring products to market faster, increase yields and enhance profits for a rapid return on investment.

Here are some frequently asked questions and answers about the Prima PRO Process Mass Spectrometer.

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A. Mass spectrometers operate by ionizing neutral sample gas molecules, and the resulting charged particle components are separated according to their molecular weight. In most commercial gas analysis mass spectrometers, ionization is achieved by bombarding the gas sample with an electron beam produced by a hot filament. To prevent collisions, the various ions are separated in a vacuum. The Prima PRO Process Mass Spectrometer is a high-performance gas analyzer based on a powerful and flexible scanning magnetic sector mass spectrometer. The platform has been designed to deliver superior analytical performance with high reliability and minimal maintenance requirements.

A:  The Prima PRO Process Mass Spectrometer is recommended for:

  • Fermentation processes
  • Cell culture processes
  • Iron and steel processes
  • Ethylene, Ammonia, Hydrogen plants
  • Ethylene Oxide, Methanol, Polyethylene/Polypropylene
  • Catalysis research
  • Solvent drying

A:  Most Prima PRO process mass spectrometers are equipped with a rapid multi-stream sampler (RMS), a highly reliable device that switches sample streams without compromising the quality of the sample presented to the analyzer. Known for rock solid reliability, the RMS has proven to switch streams millions of times a year, year after year, with little or no maintenance.

A:  When compared to traditional small molecule synthesis, biological processes are very complicated. Each cell is capable of carrying out thousands of chemical reactions per second and, often times, only one reaction will result in the target molecule. How the reactions progress will be determined by a host of factors such as temperature, availability of nutrients, the amount of accumulated waste products, available oxygen, the concentration of enzymes that promote reactions and the amino acid building blocks from which proteins are made. With a simple fermentor, the growth medium is loaded following sterilization and the broth is inoculated with cells. Relatively stable sparge gas flow rates and impeller rotation rates are maintained to ensure sufficient oxygen availability throughout the medium. Once the cells start to multiply, excess heat is removed by cooling water and adjustments of pH are made using acidic and basic reagents.

Dissolved oxygen (DO2) is monitored continuously and manual assays are performed to assess cell density and substrate composition. If DO2 falls below a predetermined level, an additional shot of oxygen can be added by opening the oxygen valve for a brief period. In mammalian cell culture, a similar control methodology can be used for dissolved carbon dioxide (DCO2).

Statistical process control (SPC) tools are used to determine if the process follows an appropriate trajectory based on data that is manually entered from the lab assays. These data are also used to determine the appropriate time to harvest. Under these circumstances, batch-to-batch variation can be significant and an order of magnitude difference is not unusual. With pharmaceutical products, if the recovered active pharmaceutical ingredient (API) falls below a certain quality standard, the entire batch must be scrapped. Clearly, the Prima PRO Process Mass Spectrometer with its highly reliable online PAT provides lab personnel with the tools needed to considerably improve product quality and increase profitability.

A:  Most bacteria used for biological production require water, carbon, nitrogen and a source of energy before they can grow and divide. They also have temperature, pH and gaseous requirements. The nutrients are provided in a complex growth medium that may include a number of natural products or the media can be chemically defined for processes where natural batch-to-batch variation will present a problem. Both types of media are designed to provide the most appropriate concentration of nutrients to encourage rapid logarithmic growth until a target cell density has been achieved. At this point, the primary carbon source should be depleted to force the cells to switch to a secondary source that promotes product formation. Additional components can be included that either inhibit or induce particular metabolic pathways in order to maximize product formation and minimize accumulation of toxic byproducts.

A typical scenario for process development uses multiple benchtop bioreactors or fermentors with capacities in the one to 10 liter (L) range. Various broth recipes are paired with different cell lines to determine the most robust and potent combination. Once the best candidate groupings are selected, the process is scaled up to the 200L scale (the pilot-scale) where potential control variables are fully tested for permissible range and efficacy. In addition to pH, temperature, agitation RPM, DO2 and DCO2, potential control variables may also include:  Nutrient feed rates, Back pressure, Overlay gas composition, Sparge composition and flow rate.

To control nutrient feeds and gas compositions in real time, it is necessary to either monitor the chemistry of the broth or the gas composition of the reactor effluent in real time. Model-based advanced process control techniques can subsequently be used to make changes to these additional control variables in response to measured changes in certain output variables. Fourier Transform Near Infrared (FT-NIR) spectroscopy is a suitable technology for measuring liquid concentrations. The best technology for making gas concentration measurements is the magnetic sector mass spectrometer, a critical component of the Prima PRO Process Mass Spectrometer that significantly increases the analyzer's power and flexibility.

A:  The most frequently used methods for determining cell mass, product concentration (titer) and substrate concentration rely on the use of differential equations. These ‘state equations’ are interdependent and must be solved simultaneously to produce valid results based on initial conditions and real-time measurements. The initial conditions include initial mass of substrate (the primary carbon source), starting cell mass and broth volume. The real-time measurements include oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), respiration quotient (RQ) and measured dissolved oxygen. The outputs from the models are typically used to track progress of each batch by comparing the results with the known trajectory of a ‘Golden Batch’ which provides an ideal profile for optimum product formation. This methodology ensures that limiting conditions and/or contamination can be identified and corrected as quickly as possible.

A:  There are several emerging advanced methods for implementing Model Predictive Control (MPC), including hybrid combinations of formal (deterministic) models and Artificial Neural Networks (ANN). Essentially, the ANN models fill in the gaps where first-principle analysis fails. ANNs are so called because their structure is based on layers of interconnected nodes, similar in structure to the neurons of the brain. These networks model behavior based on historical performance. The large training data sets often show that outcomes are the result of process variables falling within a range. While it might be very difficult to derive a formal explanation of the linkage, these relationships can still be used for process control. The Prima PRO Process Mass Spectrometer enables extended analysis of the bioreactor effluent, and subsequently provides the data necessary to train these neural networks. Other mathematical modeling techniques include Principle Component Analysis (PCA) and Partial Least Squares (PLS) regression, both of which are mathematical procedures for investigating patterns and relationships in large data sets. By facilitating data compilation, the Prima PRO process mass spectrometer is a key component in successful MPC implementation.

A:  The time spent measuring the concentration of each component in the gas stream is software-configurable, enabling the trade-off between speed and precision to be varied depending on the number of sample points and the dynamic nature of each process being monitored. A typical analysis time is five seconds for the measurement of nitrogen, oxygen, argon and carbon dioxide plus an additional three seconds for the measurement of methanol and ethanol (for example). In addition, five seconds of flushing time are added, resulting in a 10-second total analysis time per stream (or 13 seconds for the inclusion of methanol and ethanol). Since the progress is slower in mammalian cell culture than in microbial fermentation, the bioreactors can be monitored more precisely and less often than the fermentors.

A:  The most important variable calculated from vent gas analysis is the Respiration Quotient (RQ). It is the function of two distinct types of activity present in both fermentation and cell culture: growth and maintenance. RQ is defined as the carbon dioxide evolution rate (CER) divided by the OUR. The Prima PRO Process Mass Spectrometer provides timely estimations of RQ that can be used to determine the current metabolic activity and potentially to enable closed-loop control of certain variables, including the Glucose Feed Rate (GFR).

A:  The oxygen concentration of the sparge gas and reactor effluent that is provided by the Prima PRO process mass spectrometer are sent to a process control computer. The data are combined with flow measurements and batch volume for the computation of culture oxygen uptake. The real-time calculation of the Oxygen Uptake Rate (OUR) is often used to determine the viable cell density in seed tanks, enabling determination of the appropriate time for inoculation.

Using the Prima PRO Process Mass Spectrometer, the OUR measurement enables continuous kLa estimation. The oxygen mass transfer will change as the viscosity of the broth changes. The microbiologist needs to understand this relationship before moving to pilot scale. Once the dynamic nature of kLa is understood, deviations from the normal trajectory can be used to detect and correct DO2 probe drift. By monitoring changes in kLa, the Prima PRO process mass spectrometer enables personnel to more easily control agitation RPM, sparge flow and sparge oxygen concentration.

Prima PRO Process Mass Spectrometer Brochure

The use of online process analytical technology (PAT) has recently become a high-profile endeavor in the biotechnology industry. Backed by more than 30 years of gas analysis success, the Prima PRO Process Mass Spectrometer is a PAT tool designed to provide invaluable information during every stage of the process. It helps to significantly increase productivity and reduce maintenance by enabling up to 60 fermentors and bioreactors to be monitored with a single analyzer.

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