Using Chromatography Columns, Virus Filtration, and TFF in Biopharmaceutical Manufacturing

Although biopharmaceutical manufacturing covers an extensive range, it is essential that each iteration of a unit operation is in compliance with an unyielding set of operational parameters and structures to ensure that the desired outcome — a contaminant-free, viable drug appropriate for human or animal administration — is achieved.

There are three unit operations more common within the field of biopharmaceutical-manufacturing: chromatography, virus filtration, and tangential flow filtration (TFF). The successful implementation of these distinctive operations, though, needs the operator to be aware of their particular operating characteristics. For instance, chromatography necessitates constant fluid-flow rates during operations, but may have differing pumping pressures. In contrast, virus filtration necessitates constant pumping pressures, but may have varying flow rates as the filters become clogged or fouled. Furthermore, in the case of TFF, the main difficulty is to keep the pressure and flow rate constant throughout the process.

While fluid transfer occurs in any one of these particular unit operations, it is vital to know that the materials being transferred could be delicate and highly sensitive (and, in various cases, costly), indicating that it is necessary for the pumping action to be low-pulsating and low-shear, or the material will be damaged.

This article will analyze the material-handling difficulties encountered with respect to flow rate and pressure in chromatography, virus filtration, and TFF processes and explain why the design and operation of the quaternary diaphragm pump — and not other technologies like the lobe or peristaltic (hose) pump — renders it ideal for use in critical biopharmaceutical-manufacturing applications. The article will also demonstrate how the ability of the quaternary diaphragm pump to operate consistently in the growingly popular single-use applications and in a fixed stainless-steel production regime offers it the versatility to improve biopharmaceutical-manufacturing maintenance, changeover, downtime, and operational costs.

The Unit Operations

This article begins by taking a closer look at three of the very well known unit operations in the field of biopharmaceutical manufacturing.

Chromatography Columns

A typical chromatography column — made of steel, glass, or plastic — is filled with resins. The resins are compressed in a specific way, through which the feed stream product flows and purifies the product through selective adsorption to a stationary phase (resin). Chromatography columns include complex media for adsorbing the target product. These media must be handled carefully. For instance, the cost of Protein A resin could be nearly $10,000 per liter, making it incredibly important to ensure proper feeding of the resin.

In certain chromatography systems, buffer gradients are needed to realize purification of proteins. Buffers are compounds that are resistant to variations in their pH level upon adding limited amounts of bases or acids to them. For instance, the pH range of buffering salts is wide; hence, they can effectively stabilize the material’s pH level.

Often, there is a need for more than one buffer, thereby creating the need to use two or more pumps. In this application, low- and high-salt buffers are continuously mixed with varying ratios to influence the adsorption of the target molecule to the chromatography resin. This necessitates precise pumping to realize the optimal pH/conductivity conditions for particular high-resolution and adsorption purification. For instance, Buffer A and Buffer B can be used to form a gradient ranging from a low-salt to a high-salt buffer in a linear manner. Particularly, the process starts with Buffer A generating 95% of the flow, where Buffer B produces the remaining 5%. When the operation is in progress, the flow rates of Buffer A and Buffer B increase and decrease in a linear manner (90% for A and 10% for B, 75% for A and 25% for B, all the way to 5% for Buffer A and 95% for Buffer B).1

This necessitates a pumping technology with the ability to generate a highly precise flow with a high turndown ratio for potentially delivering low and high flow rates as the elution stage continues. It is also essential to reduce pump pulsation to prevent any disturbance to the packed column.2 In case the pump does not satisfy these requirements, achieving the correct buffer concentration may not be possible. Moreover, if excessive pulsation is produced by the pumping action, the buffers could be subject to experience spikes in their conductivity.

This can have an impact on the purification level of the product since the salt level in the buffer could be compromised. Furthermore, when the sample is loaded, it is normal for the back pressure of the system to increase. Pumps that do not tend to slip are advantageous in these situations as their flow rates stay constant and the linear velocity remains stable. In simple terms, the flow rate of a pump with minimal slip can be more easily controlled since it will require only incremental adjustments to the speed of the pump (measured in RPMs).

Virus Filtration

Virus-filtration systems are used in biopharmaceutical manufacturing to guarantee the safety and viability of the drugs produced by removing potential contaminants from products that are developed through cell cultures. While chromatography is characterized by variable pressures and constant flow rates, the functioning of virus-filtration systems is in contrast — a majority of the virus-filter applications involve constant pressures with variable flows. Put differently, it might be necessary to increase and decrease the flow rate or speed of the operation to maintain a constant pressure.

As discussed above, the flows vary due to the clogging of the virus filter. A majority of the typical virus-filtration systems work at a constant pressure, for instance, 2 bar (29 psi), owing to the nature of the tight pores in the filtering medium; however, there will be a decrease in the flow rates as the pores of the filter become fouled. If this is the case, then the flow rate will not decrease in a linear manner, thereby adversely affecting the filter performance, product yield, and overall quality.

Certain virus filters are designed with a flux-decay capacity of nearly 90% of the starting flux rate, necessitating the use of a pump with a high turndown ratio as well as the ability to produce minimal pulsation in the pumped fluid. Assessment of viral clearance approaches needs demonstration of the equivalence of scalability from bench to manufacturing scale and vice versa.3 In spiking studies for virus-filtration, a pressure vessel including a small surface area is used. This vessel can measure as small as 5 cm2 and can require a pump with a low-shear and low-pulsation operation if commercial-scale production levels are to reflect the small-scale studies. Under these conditions, the use of low-pulsing pumps can guarantee that pressure conditions at the time of validation of the specific filter do not fall outside the validated range.

Tangential Flow Filtration (TFF)

TFF is also called cross-flow filtration, and in this case, the flow of the biologic feed stream is tangential across the filter membrane at a positive pressure. While the flow passes over the membrane, the feed stream portion that is smaller compared to the pore size of the membrane passes through the membrane. This is in contrast to the so-called normal-flow filtration (NFF), or “dead-end” filtration, where the feed flows completely through the filter membrane and the pore size determines the portion of the feed that is permitted to pass through and the portion that will remain trapped in the filter membrane.

TFF varies from NFF in biologic applications since the fluid’s tangential motion across the membrane prevents the build-up of molecules from a compact gel layer on the membrane surface. This operational mode indicates that a TFF process can take place continuously with comparatively high protein concentrations with less binding or fouling of the filter.

It is necessary to successfully control two variables to scale up a TFF process. Recirculation (cross-flow) is essential for reducing the formation of the gel layer and pressure is required as the driving force to push the permeate through the membrane. It is important for the recirculation rate to work in coordination with the pressure (called the trans-membrane pressure, or TMP, or the average amount of pressure applied to the membrane). It is crucial to maintain a constant TMP since if it is very high, it can lead to gel-layer formation that cannot be eliminated by recirculation, and if it is very low, it leads to low flux that will affect process efficiency.

In this circumstance, pumps delivering low-pulsation flow characteristics will operate in a more reliable manner by reducing the fluctuation in the variables. Therefore, when the functional design of chromatography columns, virus-filtration systems, and TFF systems are taken into consideration, the common factor in ensuring reliable, efficient, cost-effective operation is to identify and use a pump technology that has the potential to produce low-pulsation as well as low-shear operation in spite of varying pumping pressures and flow rates.


  2. L. Hagel, G. Jagschies and G. Sofer, Handbook of Process Chromatography: Development, Manufacturing, Validation and Economics, 1997
  3. H. Aranha and S. Forbes, “Viral Clearance Strategies for Biopharmaceutical Safety” Pharmaceutical Technology, June 2001

This information has been sourced, reviewed and adapted from materials provided by Quattroflow.

For more information on this source, please visit Quattroflow.


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