Behind the Scenes with Buffers

Print 12 March 2015
Cynthia Challener, PhD / BioPharm International

The therapeutic efficacy of protein-based drugs depends in large part on their structure. Unfortunately, many proteins are highly sensitive to pH and other environmental factors and therefore are unstable unless maintained under certain conditions. To maintain a viable environment, different buffer solutions are required for nearly all downstream biopharmaceutical processing steps, including filtration, capture, and chromatography operations. Managing the preparation of large quantities of various solutions can be a challenge, particularly as upstream titers continue to increase. The development of in-line dilution systems and appropriate single-use equipment has been important for addressing many buffer handling issues.

Why buffers?

A buffer solution is comprised of a weak conjugate acid-base pair, for example, a weak acid and its conjugate base, or a weak base and its conjugate acid. Small quantities of other acids or bases are neutralized when added to the solution, and as a result, the solution resists changes in pH. Solutions with stable pH are required for the processing of protein-based biologic APIs because of their sensitivity to changes in pH. The stable pH range and capacity (the amount of acid/base that can be added without a change in pH) of a buffer are determined by the conjugate acid-base pair.

Most purification steps for downstream processing of biologics require buffers to equilibrate, condition, wash, elute, or concentrate the product. They are used in product isolation and purification steps to facilitate protein capture and polishing and also in the filtration process for the removal of viruses or depyrogenation. Aqueous solutions are also required for cleaning and disinfecting downstream processing equipment, such as chromatography matrices and membrane filtration systems. The main equipment involved in downstream processing with buffers includes mixers for buffer preparation, as well as equipment for purification, including packed-bed chromatography systems, fluidized beds, ultrafiltration and diafiltration membranes, microfiltration membranes, depth filtration cartridges, and virus-removal systems.

Buffer Challenges

While buffers make it possible to process protein-based APIs, the need to use them does present some difficulties for biopharmaceutical manufacturing. Buffers are the largest constituents by volume in the downstream processing of biotherapeutics, according to Kimo Sanderson, vice-president of client services at Asahi Kasei Bioprocess. Each process typically requires a specialized buffer composition. Furthermore, buffer solutions used in biopharmaceutical manufacturing are very dilute, and large quantities are required. “The main operational challenge regarding buffers in downstream processing is make-up and storage of the buffers. A single 1000-L cell-culture harvest may require a total of 15,000-20,000 L of aqueous buffers to extract and purify the therapeutic protein so that it meets the drug substance specifications. Thus, buffer preparation and storage are significant and critical operations that must be carefully planned into the production schedule,” says Scott Lorimer, a scientist with Patheon Biologics, a Netherlands operation. There is, not surprisingly, a drive to reduce the labor and space (and hence, costs) involved in buffer preparation and handling.

Importance of Upstream Processes

One of the most significant advances in biopharmaceutical manufacturing that has impacted downstream processing actually occurred upstream and relates to significant improvements in titers. “This technology advance in cell-line productivity has impacted downstream processing capacity, where buffer preparation can be rate-limiting to the output of a therapeutic-protein or monoclonal-antibody facility,” Lorimer notes.

Buffer use is also impacted by the upstream production method, with microbial fermentation and cell culture presenting different downstream processing requirements. Generally, according to Lorimer, purification of microbial fermentation products requires a greater number of unit operations because there are more insoluble solids and impurities. “A typical downstream process for a microbial product requires steps for host-cell disruption, removal of the cell debris, solubilization of the product, and removal of the greater load of intracellular impurities from the product stream. In some cases, microbial purification processes also require more aggressive solutions, such as urea or guanidine hydrochloride, in order to solubilize inclusion bodies,” he explains. In addition, because biotherapeutics produced via fermentation are typically smaller proteins, they often require smaller bead chromatography media and higher pressures for purification than are used for the larger molecules produced via cell-culture processes. “For some of these smaller proteins, the use of ion-exchange chromatography in aqueous environments and reverse-phase chromatography in dual aqueous/organic environments is also possible without denaturing the protein, while the larger size of mammalian cell-derived proteins allows for only aqueous elutions. These options must be considered when developing buffer management plans,” Sanderson says.

On the other hand, mammalian cell-culture harvests are generally “cleaner” than microbial fermentations with much reduced biomass, bioburden, and with fewer cellular impurities once cell separation has been completed. However, Lorimer points out that cell cultures have a theoretical risk of carrying viruses within the host cell, and therefore, downstream processes for cell-culture biopharmaceuticals must be designed to include robust viral inactivation and virus removal.

The In-Line Dilution Solution

Batch-mode buffer production involves off-line quality control measures to ensure buffer accuracy and large tanks that are costly to clean and occupy valuable space within pilot plants and manufacturing facilities, according to Sanderson. He also observes that even in newer single-use facilities, tank cleaning is no longer an issue, yet the buffers themselves are still compounded in a labor-intensive manual fashion. “These obstacles create bottlenecks that lengthen processing times, particularly when high-protein titers are involved. As a result, a new paradigm has emerged in the form of in-line buffer dilution, which has been inspired by the need for greater functionality packaged within a smaller form factor compared with legacy systems,” Sanderson says.

In-line buffer dilution involves the use of concentrated salt solutions that are diluted with water, pH-adjusted, and mixed as they are sent to the downstream processing step. Because concentrated solutions are used, much smaller storage equipment and less space are required.

It should be noted, however, that in-line buffer mixing can be challenging, because the buffer solutions must be well mixed and meet tight specifications for pH, temperature, and other critical parameters when they are delivered to the process step. Because of this, strict control over the solutions is necessary. The consequences of poor mixing can be significant and range from reduced process performance to the production of out-of-specification products.

If the proper controls are in place, however, such innovative buffer blending technology helps the production floor generate more precise, diluted buffers on-demand, according to Sanderson. The in-line buffer system from Asahi Kasei Bioprocess utilizes process analytical technology to simultaneously control the pH and conductivity of buffers generated from up to 20X salt solution concentrates and ensure that they continually meet the required specifications.

Impact on Adoption of Single-Use Technologies

The introduction of in-line buffer dilution has tied in well with the growing adoption of single-use technology in biomanufacturing. Two recent advancements in the industry are single-use tangential flow filtration skids and single-use prepacked chromatography columns, according to Derek Masser, director of sales-life sciences at ASI. With respect to buffer preparation and storage, however, there are practical size limits. The largest volume of a disposable bag is approximately 3000 L, which is not sufficient for conventional dilute buffer preparation. If concentrated buffers are used, on the other hand, disposable bags are an ideal solution.

In fact, single-use disposable containers can facilitate the rapid preparation of several buffers in the same room without risk of cross-contamination between the buffers, according to Lorimer. “With single-use equipment, several downstream buffers can be prepared quickly and cleanly for immediate use in biomanufacturing processes. And because single-use disposable systems generally do not require steam-in-place or clean-in-place utilities, the footprint and capital cost of buffer preparation can be reduced even further than what is possible by using concentrated solutions and in-line dilution,” he says.

However, temperature control remains an issue in downstream processing, according to Masser. To help address this issue, ASI recently introduced two new single-use technologies, a single-use heat exchanger for maintaining a constant temperature during various downstream processing steps, and a single-use bag and container combination for the freezing/thawing of biopharmaceutical fluids in commercial equipment.

Masser also believes that while recent technology developments, such as in-line buffer dilution and various single-use purification systems, have improved yields in downstream processing, true advancements will come only when downstream steps can move away from batch to continuous processing.

Instability Issues Remain

Another challenge facing biopharmaceutical manufacturers is the instability of some process intermediates in aqueous buffer solutions. For some unstable products, extended exposure to buffer solutions can lead to degradation, so hold times between unit operations must be limited to avoid variable product recovery, according to Lorimer. For others, temperature is the issue, and thus cold processing at 2-8 °C may be required. For product intermediates that undergo oxidation, nitrogen-blanketing of buffers and downstream unit operations is necessary to prevent product depletion and the production of unwanted impurities. All of these requirements add cost and complexity to downstream processing. Lorimer does note that there are some protein-stabilizing technologies that have typically been used for final formulation development, but have potential for stabilization of process intermediates and are currently under investigation in these applications.

Changing Landscape

Other factors affecting downstream processing and the use of buffers in biopharmaceutical manufacturing relate to the overall changes in the pharmaceutical industry. “The changing landscape of the biopharmaceutical industry has had the greatest impact, as the decline in blockbuster drugs has triggered a shift towards orphan drugs, biosimilars, and lower-volume manufacturing operations tailored to the demands of smaller patient populations or local markets,” says Sanderson. “In addition, as novel drug candidates for targeted delivery are becoming more potent, lower product volumes, and thus, smaller batch sizes, are sufficient. Lastly, new technologies are required, particularly in crowded manufacturing suites, as manufacturers embrace new approaches rooted in increased efficiency, lean manufacturing, and continuous processing.” He concludes, therefore, that the challenge lies in implementing space-saving, yet powerful equipment capable of creating a broad range of buffers simultaneously and in different capacities without any drop in quality or accuracy.

About the Author
Cythia A. Challener, PhD, is a contributing editor to BioPharm International.

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