Q&A from our webinar: Fast and efficient viral vaccines purification by sucrose density gradient

In this blog, we answer questions we received during our webinar "Fast and efficient viral vaccines purification by sucrose density gradient". 

You mention the process connection for refractometer can be done to the outlet hose of the centrifuge through a small flow cell. What is the smallest volume or flow that can be measured with that flow cell, and does it meet requirements for pharmaceutical manufacturing?

Connection is done directly to the discharge line of the centrifuge with the Pharma Mini Flow Cell (PMFC), for example through a medical hose of 4.8 mm/0.189 inches (inner diameter), 8 mm/0.314 (outer diameter). The PMFC was designed to meet the recommended flow velocity of 1.5 m/s or 4.92 feet/s over the refractometer’s prism at all times. It has a sample volume as small as of 1.2 ml.

The PMFC meets requirements for pharmaceutical manufacturing just like our pharma refractometer model. The wetted parts materials for the Pharma Mini Flow Cell are AISI 316 stainless steel. Its wetted parts are electropolished to an inner roughness of 0.4μm (equivalent to 15 μ inch). EPDM material (ethylene propylene diene monomer) for the O-ring sealing, and of course all connection fittings are 3-A Sanitary Standards.

What is the refractometer’s response time and does the length of the unloading hose from the centrifuge make any difference?

The speed of response for the Vaisala K-PATENTS Pharma Refractometer is 1 second undamped. However, damping time selectable up to 5 minutes. The length of the hose is a common question from customers, since in this application the measurement and control are critical for identifying, as quickly as possible, the virus-rich fraction and to maximize productivity. For our Pharma Refractometer, the length of the hose does not make any difference because the measurement can be delayed to the required digit with our software.

Could you describe more how to do the verification for the instrument? Can it be done on site?

The refractometer is shipped factory calibrated for refractive index; it does not require any re-calibration as the measurement does not drift over time. However, GMP guidelines require a periodical verification further defined by a company’s procedures and quality system.

Verification of the refractometer can easily be done on site by using a sample holder supplied by Vaisala and a set of five Cargille standard refractive index liquids that cover the full range. These are the same liquids as those used during calibration; therefore, the calibration can be verified and is traceable to N.I.S.T.

The verification procedure for the refractometer is build-in in the system and the user can follow a guide on the refractometer’s display for the verification procedure. A report of the verification results can be printed and signed.

We perform virus purification by cesium chloride (CsCl) density gradient. Is the Vaisala K-PATENTS refractometer suitable for this type of purification?

The Vaisala K-PATENTS Pharma Refractometer is suitable for measuring density gradients regardless of the type of solution used for the purification, provided that the differences in density is large enough to be detected (typically +/- 0.1% bw). The main objective is to measure changes in concentration in order to detect different gradients and isolate the one containing the particle of interest, in this case the virus. 

One consideration is the concentration range of the solution used and thus refractive index range required during purification. As a standard, the refractive Index (nD) range of the refractometer is 1.3200 – 1.5300, which corresponds to 0-100 Brix. Aqueous solutions of cesium chloride do tend to have slightly lower refractive index values compared to sucrose solution. However, that is not a limitation for the measurement. In addition, special prism materials are available for extending the measurement range.

Generally speaking, when it's useful to measure Brix as opposed to percentage by weight? How many measurement units are selectable?

Typically, Brix is used when sugar solutions are used as the medium for the gradient in virus purifications. Brix is a well-known method for measuring sugar content in aqueous solutions and traditionally is measured by a refractometer. However, the it is the customer’s choice. Refractive index measurements by the Vaisala refractometer can be easily configured to show Brix, % by weight, volume, density, or any other unit preferred by the customer.

What's the influence of viscosity in Brix and concentration measurements?

Viscosity, just like density, varies depending on the concentration of the solution. When a density gradient is prepared by using solutions of different strengths (e.g. sucrose solutions), typically a gradient is obtained in which not only the density is different but also the viscosity. Some solutions, however, can have nearly the same densities among different concentrations, but have very different viscosities. In this case, consider using a viscosity gradient instead to achieve an efficient separation of viral particles.

It is important to keep in mind is that the gradient is formed by solutions of different concentrations, and thus the choice of using a viscosity gradient is not a limitation for the Vaisala K-PATENTS pharma refractometer. Viscosity measurements can be considered for the fractionation and isolation of virus particles whereas refractive index measurements in virus purification application. Refractive index measurements indicate changes in total dissolved solids in the mixture, which helps to reliably monitor the gradient during centrifuge unloading and to safely isolate the rich-virus fraction. Vaisala K-PATENTS Pharma Refractometer can be used also with very high viscose solutions.

What is the maximum registration time for data?

Data logging with the Vaisala Multi-channel User Interface and Compact User Interface is possible up to a full week. Measurement for refractive index, temperature, concentration as well as some parameters for diagnosis are recorded once per second. If the Vaisala K-PATENTS stand-alone refractometer is connected directly to customer´s process control system, then the maximum data registration time depends on the system capability. The refractometer makes a measurement every second, so the data logging interval can be adjusted from one second upwards in the system.

You mentioned that the centrifuge typically has three phases of Brix concentration. What are the typical ranges in Brix for each phase? And what is the accuracy of the Vaisala Pharma PR-43-PC Refractometer?

Typical Brix concentrations in the production of influenza vaccines (sucrose gradient) are between 0 and 60 Brix. However, this may be specific for each virus and purification protocol. The Vaisala refractometer measures the full Brix range of 0-100 with the same accuracy of +/- 0.1 Brix. Also, an accuracy of 0.05-0-02 Brix is possible with the high-accuracy (HAC) version for some applications.

Is there any difference in sensor used for lab and process?

The same refractometer unit can be used at all scales in pharmaceuticals development and manufacturing. The only thing that varies is the process connection. The compact design of the refractometer allows for easy use in the laboratory with a laboratory cuvette (LTC), and in pilot with pharma mini flow cell (PMFC) for low volumes or any other sanitary process connection suitable for 2.5 in pipes or less.

Is the refractometer a suitable measurement tool for other viruses beyond influenza?

Yes. Refractometer usage does not depend on the virus to be purified, but on the gradient solution used in the purification. In addition to the influenza vaccine manufacturing, the refractometer can be used for example when making rabies vaccine or hepatitis vaccine. Concentration range in which the virus rich product fraction is to be found and collected tends to be specified according to the virus.

Explore all refractometer applications in pharmaceutical manufacturing.