A Closer Look at Automated In-Line Dilution

Published on: 
BioPharm International, BioPharm International-10-01-2009, Volume 22, Issue 10
Pages: 40–45

Automated in-line dilution can help solve capacity, financial, and quality concerns that biopharmaceutical manufacturing plants may be facing.


Automated in-line dilution is an increasingly popular technology in the biopharmaceutical industry. In-line dilution is a process that can help solve capacity, financial, and quality concerns that biopharmaceutical manufacturing plants may be facing regarding process solution preparation and delivery. This technology has several applications in biopharmaceutical manufacturing, such as purification processes, chromatography systems, solvent adjustment, pH adjustment, and cleaning systems. The fundamental aspects of automated in-process dilution systems are discussed, including engineering considerations, equipment components, process materials, operation, maintenance, and quality considerations.

Automated in-line dilution is a process in which two liquid streams are brought together in a controlled fashion to meet a target diluted solution concentration. A dilution ratio of 10:1 or more often can be achieved using current equipment designs. This typically allows for concentrates of up to 10x concentration to be used as starting material. The maximum dilution ratio is limited by both equipment constraints and the properties of the concentrated solution. Much larger dilutions can be obtained by placing multiple in-line dilution processes in series.


The equipment used for an in-line dilution process is compact and usually portable. The most basic equipment would include only one module and only perform one process step at a time, meaning that only two inlet streams are combined to make an intermediate or final product. If a second process step such as addition of another solution or adjustment of another parameter (e.g., pH) is desired, additional modules can be added to the equipment train. Multiple skids also can be placed in series to accomplish this task. An intermediate is the output of any individual in-line dilution module or skid. The intermediate is then directed to the inlet of the subsequent module to perform the next processing step. Figure 1 illustrates a process in which multiple dilutions or processing steps can be performed.

Figure 1

Automation of the process allows for the final product solution to be manufactured "just in time." The small portable equipment is capable of delivering the final product at the point of use. Figure 2 shows an in-line dilution skid.

Figure 2


The use of automated in-line dilution systems provides very significant advantages to biopharmaceutical manufacturing. One of the many concerns in biopharmaceutical facilities is capacity; biopharmaceutical companies are now obtaining much higher fermentation and cell culture yields than in the past, leading to capacity shortages in downstream processing equipment. Another challenge is manufacturing at large scale. Manufacturing 10,000-L batches of process solutions in large tanks is inherently difficult. Making a 1-L solution in a laboratory can be done very precisely using analytical instruments that are calibrated at milligram sensitivity. In contrast, making a 10,000-L solution requires load cells or level probe technology with much less precision. Mixing at large scale necessitates mixing studies and process validation to ensure that the mixing process is reliable and repeatable.

In-line dilution technology provides significant advantages compared to traditional large-scale processes because the mixing and preparation was actually being done at a small scale; compare the holdup volume of the in-line dilution skid versus a 10,000-L buffer prep tank. In addition, in-line dilution processes can incorporate feedback control with mixing to achieve highly accurate solution concentrations.

As stated previously, a process that was originally designed for a 10,000-L batch of process solution may now require twice as much solution because of increased yields. The manufacturing process now needs to make two 10,000-L batches in the preparation vessel and transfer each batch to a 20,000-L storage vessel. Is there a 20,000-L vessel available? Is there room to install a tank this size? Figure 3 illustrates how in-line dilution can solve this large-scale problem by using a small 2,000-L tank of concentrate compared to the large 10,000-L and 20,000-L tanks needed in the previous example.

Figure 3

It is not uncommon for organizations to hesitate or be uncertain about implementing new technologies. There is comfort with traditional processes where there are known failures and a general knowledge of how they are addressed. In addition to addressing new process methods, biopharmaceutical manufacturers must consider recent US Food and Drug Administration and International Conference on Harmonization (ICH) guidelines.1–3 These guidances urge companies to use process analytical technologies (PAT) in support of quality by design (QbD) and continuous improvement initiatives. The thought of incorporating PAT adds another level of complexity to adopting new process methodologies.


Equipment for automated in-line dilution accomplishes the following process steps: concentrated solution is combined with diluent solution, the combined liquids are mixed, solution concentrations may be adjusted depending on the system components, and the final desired solution is delivered for its intended use. Engineering considerations needed to successfully accomplish these steps are discussed in the following sections.

Equipment Design

There are two fundamental equipment designs for in-line dilution processes. Several variations of the basic designs are commercially available. These variations differ primarily in the method used to generate fluid flow and measure the streams. Examples of equipment designs include:

Pressure-controlled. Fluid flow is controlled by changing the pressure of the inlet streams. For example, a beverage machine uses pressurized carbon dioxide (CO2) to mix concentrated syrup with water to produce soda with the appropriate ratio of syrup to water (Figure 4).

Figure 4

Mass-controlled. Fluid flow is controlled by measuring the mass flow rate.

Volumetric. Fluid flow is controlled by precision metering pumps.


Parametric parameter feedback. Feedback control is accomplished by measuring the output of selected solution attributes.

Parametric parameter feedback with dynamic blending. Feedback control is accomplished by measuring the output of select solution attributes and blending is performed in a continuously mixed blending module.

Designs such as pressure-controlled, mass-controlled, or other mechanically based controls are not considered QbD or PAT because they do not directly identify, monitor, and control a critical process parameter (CPP). The CPP in buffer blending, for example, is the chemical make-up of the buffer (molarity and pH), not its mass, pressure, or volume. By controlling the CPP, one can eliminate the impact of out-of-specification (OOS) concentrates and the extreme difficulty and cost of trying to make nearly perfect concentrates with mass, pressure, or volume blending designs. Designs that use a blending module with PAT and feedback controls make it possible for the dilution process to decouple the variability of the final product from the variability of the concentrate.4

Equipment Components

There are three basic equipment components that comprise in-line dilution equipment. These include piping, fluid flow devices, and mixing devices. Control instrumentation includes mass flow meters and analytical instruments such as pH, conductivity, or near-infrared (NIR) instrumentation. A programmable logic controller (PLC) integrates the operation and control of all components.

Piping and Material Compatibility

Like most equipment in the biopharmaceutical industry, in-line dilution skids are primarily constructed of 316 L stainless steel tubing connected by sanitary fittings and ethylene propylene diene monomer (EPDM) or silicone gaskets. EPDM and silicone are widely used in the pharmaceutical industry because of their favorable chemical and temperature resistance and spongy-elastic properties. Gaskets are also available in Viton, PTFE, and many other compounds. The material chosen must be compatible with the process liquids and must pass US Pharmacopeia (USP) Class VI testing. Other metals such as Hastelloy or AL6XN can be substituted for 316 L stainless steel in applications where corrosive liquids are handled; these alternate metals have different elemental ratios and different compatibility properties. Product concentrates may require special considerations for compatibility; after dilution, standard product contact materials should be acceptable.

Fluid Flow Devices

Fluid flow is accomplished by the following:

Compressed air. In pressure flow designs, fluid flow is accomplished by using compressed gas to pressurize the initial source of the concentrate and diluent. Since fluids flow from high to low pressure, the fluids under pressure have the ability to flow through the process because the outlet of the process has little or no pressure.

Positive displacement rotary lobe pumps. In mass-flow or blending module designs, rotary lobe pumps can be used. Rotary lobe pumps consist of two rotating lobes that are similar to gears. The inlet side of the pump allows liquid to flow in. The liquid is then forced through the outlet at a greater pressure than the inlet side, which results in flow (Figure 5).

Figure 5

Diaphragm metering pumps. Diaphragm metering pumps may be used in metering and blending module designs. Metering pumps move precise volumes of liquid per revolution to provide accurate flow rates. The amount of flow is changed by either changing the stroke length or by adjusting the cycle frequency.

Centrifugal pumps. A centrifugal pump uses an impeller to draw in liquid at the center and force the liquid outward as it spins. The liquid gains energy (increased speed) as it is forced outward by the impeller. As the liquid exits the pump it slows down again, but now contains energy in the form of increased pressure, resulting in flow (Figure 6).

Figure 6

Mixing Devices

Mixing devices include:

Static mixer. Static mixers are pipes with baffling inside to force a turbulent fluid flow condition. The turbulence causes the liquid to mix.

Blending module. A blending module is a small continuous mixing chamber that is located between the inlet and outlet streams. The chamber could consist of a small tank or a large pipe (Figure 7).

Figure 7

Control Instrumentation

Control instrumentation monitors the control parameters for solution input to the system, and monitors parameters measuring the attributes of the diluted solution. The following are used:

Mass flow meters. A mass flow meter uses the Coriolis effect to measure the changes in vibration of a pipe as mass flows through it. Because there are no moving parts, this results in a reliable instrument that does not wear out and is not likely to drift out of calibration (Figure 8).

Figure 8

Conductivity sensors. Conductivity is impacted by the amount and type of ionic chemicals such as metals and salts in the solution. It is also impacted by solution temperature; therefore, adjustments must be made to compensate for this effect (Figure 9).

Figure 9

pH sensors. pH is affected by temperature, but some sensors can automatically measure and compensate for temperature. Caution must be taken for product solutions that are at the extremes of the pH scale (~0 or ~14) because some sensors cannot measure at these ranges and can be damaged by these harsh solutions, which is referred to as "pH poisoning," Figure 10).

Figure 10

Photometric sensors. Photometric sensors include UV and NIR spectrometers. This family of sensors uses specific wavelength light to determine the chemical composition of the solution. The instrument passes light through the solution to a detector on the other side, and then relays the information back to the computer for evaluation.

Programmable Logic Controller (PLC)

The PLC is a computer that contains a specific list of instructions, known as the program or code, for the equipment to follow. It also collects all of the information from the sensors on the skid and uses the data to determine if the process is operating in the acceptable limits. If not, it will attempt to correct the process to maintain it in the acceptable limits. The program alerts the operator of unsafe or out-of-specification conditions and may stop the process completely if the equipment is not able to correct the problem.


The process materials are mixed to prepare the final diluted solution of interest, as described in the following:

Process solution concentrate. In many cases, concentrates can be purchased or prepared in concentrations of 10x or more. Concentrates can be supplied in disposable flexible bags or be prepared in existing solution preparation vessels.

Diluting agent or diluent. Water is the most common diluent. The specific grade of water required depends on the specifications of the final product. Other diluting agents such as isopropyl alcohol are also possible, which must be evaluated for material compatibility and equipment safely.


The dilution skids can operate continuously for long periods of time and remain extremely reliable. The processes are usually designed with a number of precautionary measures to keep the equipment running. For example, redundant measurements can be used for pH and conductivity. This means that there are two sensors for each measurement. In the event that the primary sensor drifts out of tolerance or fails, the secondary sensor takes over and sounds an alarm to alert the operator of the event. Alarms are the second precautionary measure. Since the skids are automated, the operator does not need to attend to the process at all times. The alarms communicate any unusual conditions through messages on the screen, audible horns, or even through plant monitoring systems.


Post-validation and qualification maintenance and monitoring are important to ensure reliable operation of the equipment. The following items should be considered:

Preventive maintenance. The equipment supplier for each of the components should provide recommended maintenance procedures and the appropriate intervals to perform each procedure in order to maintain optimal working condition.

Calibration. The factors involved in determining the calibration frequency can include: critical versus non-critical measurements, the number of batches produced between calibrations, and the calibration history of the instrument.

Cleaning. The cleaning of an in-line dilution skid is accomplished by automated clean-in-place (CIP) procedures, which are much preferable to those of a traditional process involving human interaction that introduces error and subjectivity into the process. Automated processes are highly repeatable and reliable when designed and validated correctly. This again reinforces the concept of QbD.

Steam sanitization and sterilization. All components and piping must be designed properly to eliminate crevices and ensure steam will contact all surfaces. All components must also be compatible with the high temperatures of a steaming process (typically 121 °C). Any moving parts, such as pumps, must be designed with sufficient clearances to allow for the expansion of the metal as the parts increase in temperature.



In-line dilution process equipment and associated components should be validated or qualified to comply with FDA regulations. Qualification of an in-line dilution skid may include factory acceptance testing (FAT), site acceptance testing (SAT), installation qualification, and operational qualification. Failure mode and effects analyses (FMEAs) have shown that the traditional manual preparation methods cannot result in a more robust method than in-line dilution equipment, even with risk mitigation implemented.

Incoming Quality Assurance of Process Materials

The quality requirements of the product concentrates must be evaluated and appropriate for the design of the in-line dilution equipment. The acceptance specifications for processes with PAT control may not be as stringent as for processes without PAT control. In-line dilution systems designed with parametric feedback and dynamic blending are able to compensate for variations in the concentrate solution, whereas systems controlled by pressure, mass-flow, and volume cannot.

Change Management and Risk Analysis

The importance of the dilution process and its influence on subsequent manufacturing processes makes any change to materials, equipment, or process an important consideration because the dilution process is often one of the first significant steps in the manufacturing process. The effects on subsequent downstream processes and cleaning must be evaluated.

Quality Control Sampling

In-line dilution equipment with mechanical controls (mass, volume, or pressure) do not reduce quality control (QC) sampling requirements because the system does not directly control the CPPs. However, systems designed with PAT controls can significantly reduce QC sampling because CPP data are measured and recorded in real time. This helps reduce the number of deviations typically seen in the traditional process, which are mainly the result of human error. In equipment designed with PAT controls, the QC sampling is more of a confirmation that the product is acceptable, unlike the traditional manual process, where QC sampling plays a much bigger role.


Automated in-line dilution can help the biopharmaceutical industry overcome some capacity, financial, and quality challenges. This technology is compact, generates significant savings compared to traditional processes, and adds increased control to solution manufacturing processes. In-line dilution is a simple and very basic concept with relatively few equipment design options. Incorporate process analytical technology into this equipment enables superior performance that is consistent with the principles of Quality by Design and can achieve significant improvements to biopharmaceutical manufacturing and associated processes. Automated in-line dilution should be considered for new installations and for improvement to existing manufacturing processes.


The author thanks Lou Bellafiore, president of Asahi Kasei TechniKrom, for helpful discussions.

This article is based on the article "Automated In-line Dilution—A QbD Manufacturing Method" originally printed in the Journal of GXP Compliance.5

Brandon J. Patterson is a senior process engineer at Kymanox, Highland Park, IL, 847.433.2200, brandon.patterson@kymanox.com


1. US Food and Drug Administration. Pharmaceutical cGMPs for the 21st Century—A risk-based approach, Final Report. Rockville, MD; 2004 Sept.

2. US FDA. PAT—A framework for innovative pharmaceutical development, manufacturing, and quality assurance. Rockville, MD; 2004 Sept.

3. International Coference on Harmonization. Q10, Pharmaceutical quality systems. Geneva, Switzerland; 2007 May.

4. Walker J. Kick-starting PAT to achieve Quality by Design in cGMP bioprocessing. Available from: http://www.technikrom.com.

5. Patterson BJ. Automated in-line dilution—A QbD manufacturing method." J GXP Compliance. Fall 2008;12(5)20–34.