An Integrated Approach to Buffer Dilution and Storage

Genentech engineers illustrate an advanced, integrated approach to inline dilution of buffer concentrates and the use of disposable bioprocess bags for buffer storage.

By Tim Matthews, Bryan Bean, Poonam Mulherkar, and Brad Wolk, Genentech, Inc.

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Editor’s Note: Please click here for this team’s description of the use of guided wave radar (GWR) for non-invasive volume measurement in disposable bioprocess bags.

Most processing solutions in bioprocess manufacturing are very dilute. Consequently, these solutions can be prepared and stored as concentrates, thereby significantly reducing storage volume requirements. The concentrated solutions can then be accurately diluted, inline, with water and sent directly to processing steps such as chromatography or tangential flow filtration.

This strategy, inline dilution, has great potential to reduce capital expenditure of a biopharmaceutical manufacturing facility by minimizing the size and number of preparation and storage vessels. In addition, a reduction of processing solution volume allows greater implementation of single-use bioprocess bags in place of traditional stainless steel or higher alloy tanks, further reducing capital expenses. The use of disposables also eliminates the need for cleaning and steaming in between runs, while disposable bags are corrosion-resistant to chloride-containing buffers.

A bag volume limitation of approximately 2,500 L has been a hurdle for implementing buffer storage bags in very large-scale biologics manufacturing facilities1. However, using buffers concentrated by factors of 10x and greater, we have been able to extend the use of bioprocess bags to even our largest antibody manufacturing facilities.

This manuscript will discuss how Genentech developed and tested inline dilution technology for implementation in a new large-scale antibody manufacturing facility. We will also discuss the integration of disposable bag technology to the inline dilution systems. The combination of these two technologies has resulted in significant capital reduction and some operating cost advantage by using bags instead of tanks.

The Challenge


The biotechnology industry has matured quickly and the demand for biological therapeutics, particularly antibodies, has increased markedly over the past 10 years. In response to the higher production demands, biopharmaceutical manufacturing facilities have been challenged to increase protein expression titers and/or increase bioreactor production volumes. Batches exceeding 100 kg of protein are becoming more common as annual demand for antibody biologics can approach several metric tons.

As protein batch sizes increase, downstream processing buffer equipment sizes must also increase. Simply scaling up equipment size, particularly buffer preparation and storage tanks, can significantly impact capital expenditure for a new facility. Genentech was recently faced with this challenge during the construction of a new 25,000-liter bioreactor-scale antibody manufacturing facility. This led us to develop, test, and implement inline dilution systems for chromatography operations in the antibody purification train, thus reducing the size of many buffer preparation and storage vessels. We conservatively targeted buffer concentration factors up to 10x, which allowed us to reduce the size of many vessels to ≤ 2500 L and thereby use disposable bioprocess bags instead of stainless steel or higher alloy tanks.

Inline dilution is relatively simple in concept: it is the simultaneous mixing of a concentrated solution and water (or some other diluent) inside a processing line to produce a normal strength, process-ready solution. Inline dilution applications in the biopharmaceutical industry present unique challenges because the processing solutions, such as chemical buffers, often must fall within very tight specification ranges for pH, conductivity, osmolality, and temperature, which can be critical process parameters. This requires that a precise mixture of the concentrated solution and water be delivered with minimal deviation over time. Also, the solution must be well mixed prior to delivery onto a chromatography column or tangential flow filtration (TFF) system. Failure to meet precise targets could adversely impact process performance, and/or process parameters could fall outside of the validated ranges, causing a batch deviation.

Buffer Solubility Studies


Our main target application for inline dilution was chromatography buffers, so we first sought to determine the solubility limits of 30 unique processing buffer solutions. Knowledge of these limits provided our first glimpse of the potential for tank size reduction and capital savings in our future facility.

The solubility experiments were performed as follows (the results are shown in Table 1):

  • The chemicals for each specific buffer were carefully weighed and added to a 250-mL glass beaker with stir bar. A precise volume of water was added to the beaker and aggressively mixed with a stir bar at room temperature (approximately 19°C) for 30 minutes. Visual observations were made to determine if the chemicals had fully dissolved.   
  • If not fully dissolved, additional water was added after each 30-minute increment. This process was repeated until the chemicals fully dissolved. The solubility limit was noted.
  • On this first-pass study, we wanted to gather a limited set of solution physical properties such as density and viscosity to see if any particularly difficult solutions would be encountered. Temperature was measured with a VWR digital thermometer (± 1°C accuracy) and pH was measured with a benchtop pH meter. Density was measured at room temperature by weighing a 1.0-mL sample taken by a calibrated pipette and weighed on a calibrated Mettler Toledo scale. Viscosity was measured at room temperature using a Cannon-Fenske viscometer.

The results of the solubility studies were very encouraging and theoretically proved that we could reduce buffer preparation and buffer hold tank size substantially in a number of chromatography steps (Table 1). Since this study only addressed solubility limits, we had other potential challenges to address such as the filterability characteristics of the buffer concentrates and stability over time. Plus, we had to determine new pH and conductivity specifications for the preparation of concentrated buffers. These are time consuming but essential lab studies to complete. Ultimately, we decided to limit the concentration factor of chromatography buffers in our new facility to 10x, which is conservative for most of the buffers used. However, reducing tank size 10x would significantly reduce capital expenses and would still provide flexibility to concentrate further if additional capacity was needed in the future.

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