Performance optimization of continuous countercurrent tangential chromatography for antibody capture

Continuous bioprocessing holds the potential to improve product consistency, accelerate productivity, and lower cost of production. However, switching a bioprocess from traditional batch to continuous mode requires surmounting business and regulatory challenges. A key regulatory requirement for all biopharmaceuticals is virus safety, which is assured through a combination of testing and virus clearance through purification unit operations. For continuous processing, unit operations such as capture chromatography have aspects that could be impacted by a change to continuous multicolumn operation, for example, do they clear viruses as well as a traditional batch single column. In this study we evaluate how modifying chromatographic parameters including the linear velocity and resin capacity utilization could impact virus clearance in the context of moving from a single column to multicolumn operation. A Design of Experiment (DoE) approach was taken with two model monoclonal antibodies (mAbs) and two bacteriophages used as mammalian virus surrogates. The DoE enabled the identification of best and worst-case scenario for virus clearance overall. Using these best and worst-case conditions, virus clearance was tested in single column and multicolumn modes and found to be similar as measured by Log Reduction Values (LRV). The parameters identified as impactful for viral clearance in single column mode were predictive of multicolumn modes. Thus, these results support the hypothesis that the viral clearance capabilities of a multicolumn continuous Protein A system may be evaluated using an appropriately scaled-down single mode column and equipment.

Section: Introductionmentioning

confidence: 99%

Validation and optimization of viral clearance in a downstream continuous chromatography setting

Chiang

Pagkaliwangan²,

Lute

et al. 2019

“…The

γ_{b}

for Resin 2 was only 50% higher even though the binding capacity was more than 100% greater than the binding capacity of Resin 1. Reducing the loading on Resin 2 reduced the required binding time which maximized the resin productivity as determined by the CCTC model (Dutta et al, ). Even at that lower % loading, the absolute loading (35 g of mAb/L of resin) for Resin 2 was significantly higher compared with Resin 1 (21 g of mAb/L of resin).…”

Section: Resultsmentioning

confidence: 99%

“…A detailed discussion of the design of CCTC systems is presented in Dutta et al (). The two important parameters for each step are the number of stages and the ratio of the permeate/retentate flow ( γ ) in that stage, both of which affect the product yield (in the elution step) and the degree of impurity removal (in the washing and strip steps).…”

Section: Resultsmentioning

confidence: 99%

“…Continuous countercurrent tangential chromatography (CCTC) provides an attractive alternative to batch column chromatography (Dutta et al, ; Dutta, Tan, Napadensky, Zydney, & Shinkazh, ; Napadensky, Shinkazh, Teella, & Zydney, ; Shinkazh et al, ). The system performs the chromatographic operations (binding, wash, elution, strip, and equilibration) sequentially, with each step composed of multiple stages consisting of hollow fiber membranes and static mixers.…”

Section: Introductionmentioning

confidence: 99%

“…The mixed‐mode operation of CCTC also showed higher productivity than batch column chromatography (10X) while maintaining similar yield and product quality. CCTC optimization showing the trade‐off between yield, productivity, and buffer usage is discussed in detail by Dutta et al ().…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Fedorenko

Dutta

Tan

et al. 2019

Self Cite

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20-25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scaleup exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform. K E Y W O R D Santibody, continuous chromatography, protein A, purification, single-use

Fluorescent Reporter‐Based Fiber Optic Probe for Continuous Detection of Antibodies

Nandy,

Vu,

Maranholkar

et al. 2024

Measurement of antibody and antibody fusion protein concentration is vital for process development and manufacturing. Continuous, in‐line monitoring of antibody concentration could be useful in a variety of applications, such as controlling the loading of protein A columns to prevent breakthrough, monitoring bioreactor titer, and detecting leaks past ultrafiltration/diafiltration membranes. Molecule‐specific monitoring techniques are advantageous for antibody detection in cell culture fluid in the presence of complex process impurities. Here we report a continuous in‐line, real‐time IgG monitoring platform using a fiber‐optic biosensor with a replaceable sensor tip covalently functionalized with a fluor‐labeled protein consisting of a pentamer of the Z domain (a more stable form of the B domain) of protein A. The sensor demonstrates concentration‐dependent fluorescence enhancement in the presence of human IgG (0.01–0.75 g/L), with consistent signals during five runs each with 1 and 0.1 g/L IgG, and maintains its specificity in the presence of Chinese hamster ovary (CHO) cell culture fluid. A 5% breakthrough of a typical 10 g/L load would be detected in less than 20 s in a flowing stream emerging from a protein A column without prior sample preparation. This sensor platform may be suitable for monitoring IgG and fragment, crystallizable (Fc) fusion proteins in diverse upstream and downstream bioprocess applications.