Technological advancement has taken protein expression titers from concentrations measured in mg/L to those measured in g/L over just a few years (1). Annual demand for antibodies has reached several metric tons, which has spurred production of >100 kg batches of protein at a time (2). As upstream yields continue to increase, downstream purification involving process solution preparation and delivery must increase in proportion to keep pace with demand. That has placed facility and instrumentation capacity constraints front and center in the biomanufacturing industry, where many are directing resources toward eliminating the resulting bottlenecks through increased productivity and efficiency of downstream unit operations (1,2).

As shown in Photo 1, in-line buffer dilution (IBD) technology has emerged as a process analytical technology (PAT) solution to this dilemma (3). This technology is based on in-line mixing of buffer concentrates (typically 10×) with a diluent (often water) to yield a 1× product. Buffer concentrates eliminate the need for large buffer tank “farms,” which can be replaced by disposable bioprocess bags (2,4). Because a dilution ratio of 100:1 is often achieved with accuracy, the equipment used for an IBD process is compact and can prepare tens of thousands of liters of buffer per hour in a footprint <35 ft².

Photo 1:

PRODUCT FOCUS: ALL BIOLOGICS

PROCESS FOCUS: UPSTREAM AND DOWNSTREAM PROCESSING

WHO SHOULD READ: PROCESS AND PRODUCT DEVELOPMENT, VALIDATION, REGULATORY AFFAIRS, AND MANUFACTURING PERSONNEL

KEYWORDS: BUFFERS, PH, PROCESS CONTROL, CONDUCTIVITY, MASS FLOW, DESIGN SPACE, CHROMATOGRAPHY

LEVEL: INTRODUCTION

IBD technology can be greatly enhanced by engineering PAT-based feedback control into a processing system. Both economic benefits and regulatory benefits can be realized from this engineering advance. In recent years the US Food and Drug Administration (FDA) has encouraged incorporation of the quality-by-design (QbD) paradigm into manufacturing strategies by the biopharmaceutical industry (5). The basic idea of QbD is to first generate a knowledge base about the quality attributes of raw materials used in a process, then to use that knowledge to control critical operational parameters (COPs) and ensure consistent, predictable final product quality (6). A key component of QbD is defining and operating within a “design space” for each manufacturing process. Design space describes acceptable limits for COPs (4,7). And PAT provides the critical means for achieving QbD because it allows manufacturers to enhance understanding of their processes through real-time monitoring and to maintain processes within control limits (4,8).

PAT-based IBD technology can be used for accurately blending process-scale quantities of buffers with various goals in mind, so different design spaces can be constructed relative to a given process. IBD systems can be used to produce buffers

• for further downstream processing steps of target compounds (e.g., equilibration, elution, and wash buffers used in protein and/or oligonucleotide purification)

• to produce formulation buffers for final product preparation(s) (e.g., injectables such as monoclonal antibodies and vaccines)

• as an end product in themselves (e.g., eye drops, contact lens solutions, cough syrups, and biofuels).

An additional opportunity exists for in-line pH adjustment and/or dilution of in-process or final products (such as protein A eluates of purified proteins) and for pH adjustment in viral deactivation.

PAT-Based IBD Technology in a QbD Context: To understand how TechniKrom's patented IBD technology fits into the QbD paradigm, it's useful to briefly preview the basic technology platform in reference to the concept of design space (4,8,9). Photo 2 shows a sample piping and instrumentation diagram (P&ID) from the process control screen of an IBD skid.

Photo 2:

Normally, deionized water for injection (WFI) is connected to pump A (P001), and a 10× concentrate of salt buffer is connected to pump B (P002). WFI and buffer concentrate are then pumped into a mixing loop, where the buffer concentrate is diluted down to 1×. Its molarity has a specific conductivity that can be monitored and controlled by an in-line process analyzer (conductivity sensor) that interfaces with a process control feedback loop. The sensor continually monitors conductivity of the buffer blend in the mixing loop and sends an output signal to the process control feedback loop. That loop then references the current process value to a user-defined conductivity target set-point for the 1× buffer. It sends a signal to adjust the pump speeds accordingly so that the dynamic ratio of WFI to salt buffer in the mixing loop is continually adjusted toward the set-point.

When conductivity is sufficiently close to the set-point (within specification), buffer is released to a product collection vessel. When the buffer is not within specification, it is diverted to waste until the conductivity process value comes back into spec. So process conditions are always known, and no out-of-spec buffer goes to the product. In a design space, process specifications are obtained through process development and characterization of buffer critical quality attributes (CQAs) (7,8). Based on that knowledge, an acceptable range of conductivity can be established. The boundaries of that acceptable range are its upper and lower control limits (UCL, LCL), which define the design space. In an IBD system, the mean between those limits is the conductivity process set-point (8). Maintaining the buffer within specification keeps a process within its design space, so it is simultaneously validated. Once sufficient validation is shown, regulatory filing of the design space can be accomplished. In future runs, simply recording the buffer blend as it is maintained within the design space (or as it is diverted to waste when out of design space) is considered to be sufficient validation of product quality assurance (8,9). This eliminates the enormous resources invested into post-run QC analysis.

For simplicity, a binary blending configuration is presented above. However, additional levels of complexity are often incorporated such as using a pump C (P101) for buffer pH adjustment. In such a case, a pH probe would be installed in the mixing loop that interfaces with its own process control feedback loop to drive the solution pH toward a user-defined set-point. This arrangement comprises a ternary blend in which both the pH and conductivity of a buffer are adaptively and simultaneously controlled by independent process control feedback loops.

This patented technology is often engineered into the front end of a process-scale liquid chromatography (LC) skid, in which a series of buffers are made on demand and sent directly to a process column for a sequence of chromatography steps in a purification regime. In this case, UCL and LCL are defined for a design space of each buffer for each chromatography step. Only buffers that fall within their design space control limits are sent to the column. When a buffer is not within specification, it is diverted to waste until conductivity and/or pH values come back into spec. This guarantees that no out-of-spec buffer will reach the column, so it can't negatively affect the efficiency of the chromatographic profile.

The ability to monitor and control the process values of conductivity and pH in real time using PAT provides for process robustness and can help companies adaptively adjust to variability in raw material buffer feed stocks. So an IBD-controlled process can provide consistent and predictable quality assurance, with quality built in to the process itself and operating within the design space provides on-the-spot validation (7,8). Optimally, when the design phase of a PAT-based IBD process includes application of six sigma and failure mode and effect analysis (FMEA), the full economic and regulatory benefits can be realized (10).

For our study, we chose a statement from the FDA's PAT guidance as a standard for assessing the validity of using in-line buffer dilution with pH and conductivity adaptive feedback control in QbD manufacturing of bioprocess buffers:

Process monitoring and control strategies are intended to monitor the state of a process and actively manipulate it to maintain a desired state. Strategies should accommodate the attributes of input materials, the ability and reliability of process analyzers to measure critical attributes, and the achievement of process end points to ensure consistent quality of the output materials and the final product. (11)

To assess the capability of an IBD system “to monitor and manipulate the state of a process and actively manipulate it to maintain a desired state” (11), we performed two buffer blending experiments. The first involved design and creation of a binary blend (conducted to produce 10 mM sodium phosphate and 20 mM NaCl at pH 7 from a 10× concentrate) with the goal of monitoring and controlling the conductivity of the 1× buffer within upper and lower control limits for an hour. The second experiment went a step further and was designed to create a ternary blend to produce a 1× Tris acetate buffer from 10× concentrates of Tris base and acetic acid. The goal of that experiment was to monitor and control both pH and conductivity of the 1× buffer.

Both blends also addressed the aspect of the FDA's statement that deals with “accommodating the attributes of input materials” (11). The 10× concentrates were intentionally prepared in a casual manner so that exact starting concentrations would not be known. Next, experiments were designed to test “the ability and reliability of the process analyzers to measure critical attributes” by performing stability, sensitivity, and temperature variation studies on the process analyzers.