Continuous monoclonal antibody purification with capture via precipitation
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Authors
Mergy, Matthew, Ryan
Issue Date
2025-08
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemical engineering
Alternative Title
Abstract
New purification processes are required to meet the global need for high-purity, high-volume, high-dose protein therapeutics, such as monoclonal antibodies (mAbs) for the treatment of Alzheimer’s disease, high cholesterol, and infectious disease. We developed a novel intensified, continuous purification process comprised of a precipitation-based capture step followed by two flowthrough chromatography polishing steps that can meet this need by eliminating Protein A affinity chromatography, the bottleneck of the current “platform” mAb manufacturing process. We pre-process harvested cell culture fluid (HCCF) to deplete host cell DNA and remove media components that interfere with mAb precipitation, capture the mAb via precipitation using synergistic bulk precipitants (ZnCl2 and PEG), dewater and wash the precipitate slurry using hollow fiber microfiltration modules in a countercurrent flow configuration to enhance impurity removal, redissolve the washed precipitates at pH 3.5 to enable low pH viral inactivation, and employ two orthogonal flowthrough subtractive adsorbers with minimal intermediate conditioning for polishing. This process can be operated in an integrated, fully continuous mode. It addresses the volumetric throughput, process mass intensity, and cost-of-goods bottlenecks as well as the equipment and supply chain complexities associated with the platform Protein A-based capture step that currently limit global mAb manufacturing capacity. This eminently scalable process also readily accommodates increasing upstream product titers, as the precipitation-based capture step becomes more efficient as mAb concentration increases. We demonstrated precipitation-based capture with mAb HCCF feed materials from multiple industrial partners, gaining key insights which support further process development and suggest that the capture process may be platformable. During HCCF pre-processing, we deplete host cell DNA via CaCl2 precipitation to significantly reduce DNA persistence in the process, which facilitates complete redissolution at acidic pH and allows the redissolved precipitate stream to be directly applied to the first polishing step without further stream conditioning. We also pre-concentrate and diafilter the DNA-depleted HCCF in a single-pass tangential flow filtration step to remove culture media components that interfere with mAb precipitation and to standardize the precipitation feed concentration and buffer matrix, which permits the use of similar, low precipitant concentrations for quantitative precipitation (> 95%) for all mAbs studied. For the capture step, we found that the addition of CaCl2 during precipitation leads to the formation of more densely packed precipitate particles, resulting in higher sustainable flux values and better impurity removal in the dewatering and washing operations. We attained maximum sustainable conversions of 65-75% in the hollow fiber microfiltration modules, which led to host cell protein (HCP) levels as low as 11,500 ppm for redissolved precipitates. At maximum sustainable conversion conditions, we achieved yields as high as 94%, buffer consumption as low as 370 mL/g mAb, and throughput as high as 33 g mAb/m2/h (based on total membrane area of the dewatering and washing hollow fiber modules) for the precipitation capture step.
We integrated the precipitation-based capture step with two flowthrough polishing steps and demonstrated fully continuous operation of the monoclonal antibody purification process. Following precipitation capture, we redissolved the washed mAb precipitates via in-line dilution at low pH to enable loading of the first polishing step. We utilized novel flow attenuation hollow fiber ultrafiltration modules to match the flow rates of the redissolution and neutralization steps with the subsequent polishing operations, enabling fully continuous operation of the process without the use of surge tanks. For polishing, we employed two orthogonal flowthrough subtractive adsorbers and performed an in-line pH adjustment (neutralization) between the steps. We utilized the combination of a hydrophobic adsorbent (activated carbon) and a mixed-mode anion exchanger (Capto Adhere ImpRes), which results in excellent clearance of residual impurities including HCPs and aggregate species at high mAb yields. We achieved > 90% yield for each processing step and observed a significant increase in precipitation capture yield during prolonged operation at steady-state conditions, resulting in an overall purification process yield exceeding 82%. We reduced host cell protein concentrations to below 10 ppm and high molecular weight impurity levels to approximately 1% in the final purified product.
We intensified the precipitation-based capture step by increasing the precipitation feed concentration by a factor 3. We attained a maximum sustainable conversion of only 40% for the intensified process, leading to significantly lower impurity removal in the dewatering and washing steps and HCP levels of approximately 50,000 ppm for redissolved precipitates. In future implementations of the intensified precipitation capture process, filtration performance in the dewatering and washing steps must be improved to enable high-capacity flowthrough polishing chromatography operations that meet final purity targets. Intensification of the precipitation capture process resulted in significant improvements in throughput (147 g mAb/m2/h) and buffer consumption (93 mL/g mAb). We performed an environmental analysis which revealed that intensification via feed pre-concentration resulted in substantial improvements in sustainability metrics for the continuous precipitation capture process. However, additional process intensification, which can be achieved by further pre-concentration of the precipitation feed, will be required to make continuous precipitation capture competitive with continuous Protein A capture relative to environmental footprint. We also performed a simple scaling analysis of process economics which suggested that process intensification reduced the Cost of Goods for continuous precipitation capture to a value lower than continuous Protein A capture.
Description
August2025
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY