In-line process analytical technologies (PATs) hold much promise for enhancing control of biopharmaceutical manufacturing processes and, in turn, increasing process quality and reproducibility. During a January 2022 presentation, Ramsey Shanbaky (associate director of bioprocess applications at Repligen) described how his company’s CTech FlowVPX system for in-line protein concentration measurement enhances monitoring of downstream processes.
Scientists often use ultraviolet–visible spectroscopy (UV-vis) to measure protein concentration in a drug substance (DS) or product (DP). Most UV-vis instruments determine that by measuring absorbance — defined in the Beer–Lambert law as the molar attenuation coefficient (ε) × species concentration (c) × optical pathlength (l) — across a fixed pathlength, treating c as a variable. But that approach yields only one measurement, and samples must be diluted to ensure that c values fall within an instrument’s linear range.
The FlowVPX system uses variable pathlength technology (VPT) to obtain a concentration reading every 10 seconds. The instrument takes in-line readings using a Fibrette optical probe suspended from a flow cell. Samples require no dilution. The probe can be adjusted to measure pathlengths from 1 µm to 5 mm. From each reading, the system collects multiple data points. Using a proprietary Slope Spectroscopy method, those are analyzed by specialized software to generate a linear slope regression, yielding a concentration measurement. The system can measure concentrations over 300 mg/mL.
Shanbaky noted that the FlowVPX devices can be mounted directly onto process equipment or attached to a stand. To facilitate scale-up, Repligen offers flow cells for laboratory, pilot, and production processes: 3 mm, 0.5 inch, and 1 inch, respectively. Those all feature good manufacturing practice (GMP)-compliant components, and the system uses an OPC unified architecture (OPC UA) to simplify integration with process skids and other PATs. Late in 2022, Repligen will release 0.5-inch and 1-inch single-use formats based on polyphenylsulfone (PPSU).
Chromatography: Shanbaky noted that scientists from Repligen and KBI Biopharma have used the FlowVPX system to compare two protein A resins for purification of several conventional and bispecific antibodies. The team took in-line measurements of product concentration in cell-culture harvest and then process effluent, generating column-breakthrough curves in real time. That facilitated determination of the resins’ dynamic binding capacities. The team later used the system to monitor column breakthrough across affinity media, flow rates, and resin lots.
Similar methods can be used for evaluating column loading. Shanbaky described how his team passed cell-culture harvest through a FlowVPX instrument to obtain a baseline reading, then ran the material until the signal stabilized. The difference between those values yielded a real-time product-titer measurement. Based on that, the team calculated loading and elution mass.
Shanbaky demonstrated the system’s utility for ultrafiltration/diafiltration (UF/DF) using data from a comparative study of measurements obtained by Repligen’s CTech SoloVPE (at-line), FlowVPX (in-line), and FlowVPE (first-generation in-line) systems during filtration of a pilot-scale batch. An off-line A280 method served as a control.
Results tracked closely across all methods. Equally important was that in-line monitoring enhanced process control. Shanbaky discussed a UF/DF process during which the skid nearly pushed product concentration out of specifications. The FlowVPX instrument alerted operators to the problem, and they stabilized the concentration. In a second case, a skid miscalculated the product-hold volume and prematurely stopped a concentration process. The FlowVPX system helped operators to identify the problem and program the skid to reach the concentration setpoint.
Drug-Product Development: Other system applications include postfiltration product recovery, which requires careful monitoring because it involves a buffer flush that can overdilute a drug substance. Real-time measurements help operators to ensure that protein concentrations remain within specified ranges. FlowVPX technology similarly can monitor dilution of a bulk drug substance for drug-product formulation.
Questions and Answers
How might FlowVPX system users approach validation? Typically, users implement the system for a specific process in a development laboratory, comparing its performance with that of a standard off-line instrument. Next, analysts validate the system’s software and connectivity with a process skid. Finally, analysts validate the system’s performance in a complete process, including steps for cleaning and disposal.
Can the FlowVPX system handle high-viscosity solutions during UF/DF? Yes, but operators should consider whether pumps and other equipment could form bubbles in the process solution. If so, operators should let filtration reach its setpoint, then let the material recirculate and homogenize.
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