A¬†strong connection between turbidity and total suspended solids (TSS) has been linked in the past to measuring well defined particles in processes. Optical density probes have seen wide adoption¬†in the biotechnology industry for¬†monitoring cell growth within a¬†bioreactor, whereas in-line turbidity¬†sensors have been used to monitor filter¬†performance. Turbidity measurements¬†offer a rapid quantification of suspended
solids but have not been used in the¬†biotechnology industry for¬†chromatographic resins. In this study,¬†turbidity measured with equipment¬†developed by PendoTECH was used with¬†novel continuous chromatography¬†technology developed by Chromatan for
accurate measurements of¬†chromatography resin slurry¬†concentrations.
Continuous countercurrent tangential¬†chromatography (CCTC), developed by¬†Chromatan Corporation, has overcome¬†many limitations of batch columns without
the drawbacks of multicolumn systems (1).¬†The CCTC platform is a true moving-bed¬†technology that runs at steady state and¬†eliminates the use of columns. The¬†system‚Äôs novel design enables short¬†residence times, consistent product¬†concentration and quality, and easier¬†implementation of advanced process¬†analytical technology (PAT) and process¬†control. In addition, CCTC is uniquely¬†suited for processing sensitive molecules¬†because of its ability to control buffer and¬†micromixing conditions independently for¬†all chromatographic steps. That eliminates¬†product-related concentration and buffer¬†gradients that always accompany column-based separations.
The patented CCTC system includes¬†all of the traditional chromatographic¬†process steps: binding, washing, elution,¬†stripping, and equilibration. Those are¬†comparable with traditional batch¬†chromatography, but they are conducted¬†simultaneously on a moving slurry rather¬†than with the step-wise batch approach¬†of a packed column (Figure 1). Each step¬†comprises a cascade of stages that¬†consist of a single static mixer connected
in series to a hollow-fiber membrane.¬†Countercurrent configuration of these¬†stages enables enhanced impurity¬†removal and higher yield for each¬†chromatographic step. The static mixers¬†provide sufficient residence times for¬†adsorption/desorption to occur. The¬†microporous hollow-fiber membranes¬†retain the large resin particles while
allowing all dissolved compounds¬†(proteins and salts) to diffuse across the¬†membrane into the permeate.
The CCTC system has been shown to¬†¬†operate continuously 5-15√ó higher¬†productivity than with batch columns,¬†with the product eluting at steady state.¬†The steady-state ‚Äúpeak-free‚ÄĚ product¬†stream can be measured in-line for¬†product quality attributes. The steady-state nature of the process also enables¬†seamless integration with other
continuous/in-line unit operations. More¬†details are provided in Dutta et al. (2).
As a consequence of these benefits,¬†The National Institutes of Health (NIH)¬†has funded a US$1.75 million Fast-Track¬†Phase 2 SBIR to support the integration¬†of CCTC and a perfusion bioreactor into a¬†single steady-state bioproduction¬†platform. The FDA also has financed a¬†$2.5 million contract to develop and
commercialize a fully integrated¬†continuous downstream process that¬†includes capture, intermediate, and¬†polishing CCTC steps for antibody¬†purification (Figure 2).
As a major strategic deliverable, both¬†integration project s require significant
developments of in-line process¬†analytical technologies (PAT) and¬†continuous process monitoring. Single-use sensors for process parameters such¬†as pH, conductivity, flow rate, and¬†pressure have been developed and¬†tested by the industry. However, because
of the true moving-bed nature of CCTC, it¬†also has become necessary to develop a
robust in-line measurement for accurate¬†determination of chromatography resin
concentration in the CCTC slurry
PendoTECH offers in-line single-use UV¬†absorbance and turbidity measuring and¬†monitoring systems that collect data from¬†bioprocess fluid streams. Three single-use
polysulfone optical flowcells with¬†pathlengths of 2 mm, 5 mm, and 1 cm with¬†hose-barb process connections can be¬†installed in-line to any process stream.¬†Reusable couplers for focusing light are¬†screwed into the flowcell. Fiber-optic¬†cables connect the couplers to the light¬†source and to the detector in the compact¬†photometer. The output from the¬†photometer is a 4-20 mA signal scaled 0-2¬†AU. The systems use an LED to generate a¬†single wavelength of light, customizable¬†by PendoTECH from 240 nm to 1,000 nm.¬†Additionally, PendoTECH offers a stainless¬†steel flowcell with adjustable pathlengths¬†of 0.05-2.0 mm, allowing end users to set¬†the pathlength for a linear response to the¬†process stream. Figure 3 shows the flowcell¬†offerings.¬†
Past work has demonstrated that using¬†the principles of the Beer-Lambert law, a¬†flowcell pathlength can be selected to measure a process stream accurately in its linear range (3). A 2-mm pathlength flowcell has been used with a 280-nm light source to measure the elution stream of a protein in a capture process in the CCTC. This in-line sensor provides accurate concentration and yield measurements at one-second intervals, enabling PAT.¬†
Materials and Methods
Resin Preparation: Two resin types were tested: an agarose backbone 22-¬Ķm resin¬†from Purolite; and POROS 50HQ, a¬†polystyrene divinylbenzene (PSDVB)
backbone 50-¬Ķm particle-size resin from¬†Thermo Fisher. PSDVB is used as a
standard for calibrating turbidity meters.¬†The agarose resin is significantly less
turbid. Using these materials enabled¬†investigations of the in-line turbidity at a
wide range of slurry ratios and¬†absorbances.
Stocks of both resins were exchanged¬†into 1 √ó PBS and prepared into 20%, 10%,¬†5%, and 2.5% v/v slurry ratios. The slurry¬†ratios of all the stocks were measured in¬†duplicate by gravity settling for 24 hours¬†in 12.5-mL Koehler K61141 centrifuge¬†tubes.
Offline Turbidity Meter: A Hach¬†2100Q turbidity meter, a nephelometer¬†with a 90¬į detector angle from incident¬†light and 1-inch pathlength, was¬†calibrated with Stabical Formazin primary¬†standards from 10 to 800 NTU and used¬†to measure the primary stocks of agarose¬†and PSDVB resin slurries. The 2100Q has a¬†maximum reporting turbidity of¬†1,000 NTU. Traditional nephelometers can¬†provide an accurate measurement of¬†turbidity up to 2,000 NTU depending on¬†the excitation light source and detector¬†configuration.
The agarose measured with the 2100Q¬†demonstrated a strong linear relationship¬†for the measured slurry ratios 2.5%-20%¬†v/v slurry ratio and 120-1000 NTU, as¬†shown in Figure 4, with an R2¬†of 0.99.
Although Formazin is the only¬†recognized primary standard for¬†calibrating turbidity meters because of its¬†relative stability, size distribution, and¬†consistent 90¬į light scatter, PSDVB also¬†has been used as a primary standard for¬†calibrating turbidity meters. However, the¬†PSDVB slurries saturated the detector.¬†The 2.5% slurry stock of PSDVB was¬†diluted into range, and a dilution curve¬†from 0.004% to 0.31% v/v slurry was¬†generated from 20 to 600 NTU (Figure 4).¬†ISO 7027-compliant nephelometers are
limited in this regard and provide only a¬†linear measurement that can be used for
quantification of suspended solids such¬†as resin only from 0 to 40 NTU,¬†necessitating a large dilution factor. In a¬†practical sense, however, the large¬†dilution factor could introduce a¬†significant source of error that might¬†interfere with efficient loading of resin
into the CCTC system.
PendoTECH Turbidity Meter: A test¬†solution was agitated with an overhead¬†mixer set to 400 rpm, and a peristaltic¬†pump delivered the fluid through the¬†flowcell in an upflow configuration and¬†back to the beaker. The agarose resin had¬†a linear relationship for transmittance in a¬†2-mm pathlength for the slurry ratio¬†range of 2.5-20% with an R2¬†of 0.99¬†(Figure 5). The PSDVB resin was beyond¬†the linear range for a 2-mm pathlength¬†and slurry ratios >10% v/v. Further testing¬†was performed with shorter pathlengths.
Both resins demonstrated a stable¬†equivalent reading for flowrates¬†5-40 mL/min at the same slurry ratios.
A 20% stock of PSDVB was prepared¬†and serially diluted with an identical¬†setup to the agarose resin, but with a¬†shorter pathlength. The PSDVB resin had¬†a linear relationship for transmittance in a¬†0.51-mm pathlength for the slurry ratio¬†range of 5-20% with an R2¬†of 0.99 (Figure¬†5). Additionally, the PSDVB testing¬†demonstrated that tuning into a linear¬†range is readily accomplished with the¬†PendoTECH stainless steel flowcell by¬†adjusting the pathlength. The experiment¬†also demonstrated strong sensitivity to¬†slurry ratio and can be used to quantify the exact concentration of resin being¬†circulated within the CCTC system.
Integration with CCTC
Flowcells and photometers offered by¬†PendoTECH enable in-line monitoring of¬†process streams for CCTC in both single-use and reusable formats. This study¬†demonstrates that in addition to¬†monitoring product titers, optical sensors¬†also can be used for measuring and¬†controlling CCTC resin concentrations.
The flowpath in Figure 1 shows an¬†integrated PendoTECH optical sensor at¬†the resin-loading manifold. The location¬†of the sensor helps determine resin cycle¬†time in the CCTC, which is used to¬†calculate resin volume and CCTC¬†productivity. The sensor also measures¬†outlet slurry ratio, providing in-line¬†measurement of timing to reach steady-state. Once the outlet resin concentration¬†reaches the inlet, the CCTC system¬†automatically switches the flow path into¬†a closed loop resin circulation mode
(by-passing the resin tank). With the¬†known cycle time, resin also can be¬†unloaded once the full lifetime has been¬†reached and replaced with fresh resin if¬†necessary.
Additionally, monitoring the exact¬†concentration entering the binding step¬†allows for precise resin loading. This has a¬†strong effect on output yield and product¬†quality especially for ion-exchange¬†operations. Unlike batch columns, in¬†which concentration gradients and¬†product peaks are a direct consequence¬†of the sequential batch operation, the¬†CCTC can target exact loading and¬†elution conditions throughout the
process without compromising yield or¬†product quality.
Overall, the PendoTECH single-use¬†flowcells and customizable photometers¬†open a wide range of possibilities for¬†in-line process control and PAT. Turbidity¬†and UV sensors enable the CCTC platform¬†to provide accurate measurements of¬†titer and yield in the product streams as¬†well as accurate resin concentration¬†measurements for system startup and¬†efficient resin cycling.
Chromatan and PendoTECH believe¬†that joining together to develop such¬†technologies is essential for development¬†and eventual adoption of continuous¬†single-use manufacturing at the current¬†good manufacturing practice (CGMP)¬†scale. We look forward to initiating and¬†continuing such collaborations with one¬†another, as well as with other customers¬†and suppliers.
1 Dutta AK, et al. Continuous¬†Countercurrent Tangential Chromatography¬†for Mixed Mode Post-Capture Operations in¬†Monoclonal Antibody Purification. J.¬†Chromatogr. A 1511, 2017: 37‚Äď44; doi:10.1016/j.chroma.2017.06.018.
2 Dutta AK, et al. Performance¬†Optimization of Continuous Countercurrent¬†Tangential Chromatography for Antibody¬†Capture. Biotechnol. Prog. 32(2) 2016: 430‚Äď439;
3 Renaut P, Annarelli D. Evaluation of a¬†New Single-Use UV Sensor for Protein A
Capture. BioProcess Int. 11(2) 2013: 48‚Äď51.
Corresponding author Dmitriy Fedorenko is associate director at Chromatan Corporation; firstname.lastname@example.org. Jasmine¬†Tan is associate scientist II and Oleg¬†Shinkazh is CEO at Chromatan Corporation. Dennis Annarelli, PhD, is technical manager at PendoTECH; email@example.com.