Modernizing the platform process of antibody purification for enhanced impurity clearance

Separation and Purification Technology · 2025-11-20 · 2 citations

The established platform for monoclonal antibody (mAb) purification, centered on Protein A affinity capture and subsequent polishing steps, has enabled large-scale manufacturing of therapeutic antibodies. However, persistent host cell proteins (HCPs), antibody aggregates, and product fragments remain challenging to clear, particularly as novel mAb formats and intensified upstream processes introduce higher impurity loads. In this study, we present transformative mAb purification platforms that combine novel multimodal resins operating in flow-through mode with conventional Protein A chromatography to address these limitations. Our new platform utilizes LigaGuard for the selective removal of high-risk and persistent HCPs, chromatin, and aggregates by flow-through affinity chromatography; and a size-exclusion-mixed-mode (SEMM) resin for the efficient removal of product-related low and high molecular weight impurities and residual process-related impurities. Several alternative process configurations were systematically assessed to maximize purification performance and robustness. A novel process configuration comprising an initial impurity-stripping step using LigaGuard followed by a capture step by Protein A chromatography and a polishing step using SEMM resin achieved global yields up to 86 %, with residual product-related impurities <1 %, HCPs <60 ppm, and hcDNA <10 ppb across diverse industrial feedstocks. The proteomic analyses of the polished streams confirmed the depletion of high-risk and persistent HCPs, including hydrolases and polysorbate-degrading enzymes. The use of two flow-through operations flanking a central affinity capture step enhances the performance of the Protein A resin alongside process productivity and robustness to feedstock variability. These results support a new paradigm for mAb purification, offering scalable and robust solutions for both legacy and emerging antibody modalities. • Integrating Protein A with LigaGuard and SEMM resins enhances impurity clearance. • The proposed process architectures robustly achieve up to 99.9 % monomer purity. • Proteomic analysis demonstrates reduction from >2600 to ~30 HCPs in the polished pools. • The combination of LigaGuard and Protein A resin remove cathepsins, chromatin, and lipases. • Platform met regulatory impurity thresholds while achieving high yield and productivity.

Flow-through polishing of Protein A eluates: Removal of high and low molecular weight impurities by size-exclusion-mixed-mode chromatography

Journal of Chromatography A · 2025-09-12

P. pastoris (K. phaffi) perfusion processes: evaluation of cell retention techniques and integrated purification strategies

New Biotechnology · 2025-03-01

Flow-Through Polishing of Protein a Eluates: Removal of High and Low Molecular Weight Impurities by Size-Exclusion-Mixed-Mode Chromatography

SSRN Electronic Journal · 2025-01-01

High-capacity nonwoven increases productivity of mAb purification in an all-membrane process

Separation and Purification Technology · 2025-03-19 · 1 citations

• A new multimodal anion exchange (MMA) nonwoven membrane was developed. • The MMA membrane exhibited a higher binding capacity than the counterpart resin. • The MMA membrane was used for capture of a mAb from CHO supernatant. • An all-nonwoven membrane chromatography process was developed for mAb purification. • The all-membrane process exhibited a higher production efficiency than the platform resin process. There is significant interest in alternative manufacturing processes for monoclonal antibodies (mAbs) to improve productivity and reduce cost. To identify a cost-effective and high-productivity alternative to the conventional Protein A-based mAb capture step, this study presents the development of a high-performance multimodal anion exchange (MMA) membrane utilizing N-Benzyl-N-methylethanolamine (BMEA) as the ligand which was covalently coupled to a polyglycidyl methacrylate (pGMA) UV-grafted polybutylene terephthalate (PBT) nonwoven. The DBC 10% of the MMA membrane for IgG ranged from 32.8-42.4 mg/mL at 0.5 to 5 min residence time. The membrane exhibited an excellent salt tolerance in protein binding near physiological conditions, high flow permeability and good reusability. The MMA membrane also showed a DBC 10% of 59.2 mg/mL for direct capture of a mAb from a CHO supernatant without the need for pH or conductivity adjustments, along with a recovery of 94.3%, a 1.0 log reduction value (LRV) of host cell proteins (HCPs), a 1.8 LRV for DNA, and a reduction of aggregates from 5.4% to 0.9%. This capture step was implemented in an all-membrane mAb purification process that included a viral inactivation step and two polishing steps using an anion exchange nonwoven (AEX-TEA) and a multimodal cation exchange nonwoven (MMC-MPCA). A side-by-side comparison with a platform mAb purification process using Protein A resin and two ion exchange resins showed that the all-membrane process exhibited similar impurity clearance, higher overall recovery (88.3% vs. 77.5%) and significantly reduced processing time (3.8 h vs. 13.1 h) with fewer steps as it eliminates the need for diafiltration for buffer exchange.

Modernizing the platform process of antibody purification for enhanced impurity clearance

SSRN Electronic Journal · 2025-01-01

Interlaboratory Measurement of Adeno-Associated Virus: Comparative Quantification of Full and Empty Capsids

Human Gene Therapy · 2024-12-26 · 2 citations

Recombinant adeno-associated virus (AAV) is one of the main viral vector-based gene therapy platforms. AAV is a virus consisting of a ≈25 nm diameter capsid with a ≈4.7 kb cargo capacity. Therapeutic safety and efficacy depend on the correct encapsidation of the DNA in individual virus particles, which is often characterized by the single scalar value of the ratio of full capsids with complete viral genomes to the total viral capsid number [the full-to-total (FTT) ratio]. This study reports on the interlaboratory and intertechnique variations of measurement methods for FTT among a cohort of organizations. The analytical methods used were sedimentation velocity analytical ultracentrifugation (SV-AUC) with UV/Vis and/or Rayleigh interference optics, size exclusion chromatography (SEC) with multi-angle light scattering (MALS), and tandem UV/Vis and/or refractive index, cryogenic electron microscopy, dual-wavelength ultraviolet spectrophotometry, and ELISA coupled with quantitative PCR (qPCR, dPCR, or ddPCR). FTT measurements for both AAV5 and AAV8 serotypes were similar, except for PCR-ELISA. The optical techniques (UV spectroscopy/SEC-MALS) showed <10% SD between laboratories, likely from the uniformity of existing industry protocols. AUC, while demonstrating good repeatability, had ≈25% SD interlaboratory, suggesting the need for standardized methods. PCR and ELISA had poor reproducibility due to variations in both PCR and ELISA protocols and instrumentation. The discussion presents intended future efforts to improve and harmonize these measurements to increase both the repeatability and reproducibility of AAV viral particle critical quality attributes such as FTT.

Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant

Journal of Chromatography A · 2024-02-01 · 3 citations

A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83 %. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 min, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20 % loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83 % to approximately 92-95 %.

Mixed-mode size-exclusion silica resin for polishing human antibodies in flow-through mode

Journal of Chromatography A · 2024-02-26 · 5 citations

Dextran sulfate bulk and surface-modified microfiltration membrane for simultaneous blood plasma harvesting and low-density lipoprotein removal during plasmapheresis

Journal of Membrane Science · 2024-03-12 · 5 citations