Control of antibody high and low molecular weight species by depth filtration‐based cell culture harvesting

Abstract

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36-40 g/m 2 ) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m 2 , similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing. K E Y W O R D Scell culture harvesting, depth filtration, high molecular weight species, low molecular weight species, monoclonal antibody, protein adsorption

Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy

“… 18 However, when compared to the group previous work, 19 they conclude that different depth filter media has different capacities for model protein solutions and some filters may be used to remove specific sub‐classes of HCP. A direct comparison of X0HC and X0SP 20 showed that X0SP had a 50% higher binding capacity when model protein solutions were used, crediting the improved charge functionality on the X0SP. An increased hydrophobic score was also linked to the reduction of high and low molecular weight mAb species, 21 though the filter media and filter loading were the biggest factors for this.…”

“…A direct comparison of X0HC and X0SP 20 showed that X0SP had a 50% higher binding capacity when model protein solutions were used, crediting the improved charge functionality on the X0SP. An increased hydrophobic score was also linked to the reduction of high and low molecular weight mAb species, 21 though the filter media and filter loading were the biggest factors for this.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…Centrifugation, depth filtration, and tangential flow microfiltration (TFF-MF) are the most commonly used techniques for cell harvesting and cell separation (Besnard et al, 2016;Voulgaris et al, 2016;Richardson and Walker, 2018;Carvalho et al, 2019b;Yu et al, 2019). The various clarification technologies used for the downstream processing of antibodies have been described elsewhere (Singh et al, 2016;Singh and Chollangi, 2017).…”

Applications of capillary electrophoresis for biopharmaceutical product characterization

“…For example, researchers have used reduced and non‐reduced SDS‐CGE for analyzing fractionated mAb acidic variants to identify their fragment composition [93]. Non‐reduced SDS‐CGE was also reported to quantitate low molecular weight species in mAb producing cell harvest samples [94]. Microfluidic SDS‐CGE was used as an orthogonal method to high‐performance size exclusion chromatography (HP‐SEC) for mAb fragmentation analysis and revealed more accurate molecular weight estimation than HP‐SEC [89].…”