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

Abstract

Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%–50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.

Role of harvest depth filtration in controlling product-related impurities for a bispecific antibody

“…The charge properties of the a-LC dimer were not evaluated here due to its unknown structure. With very similar pI, the BsAb1 product was strongly positive (+11.4) compared with the homodimer (+2.9) and the a-LC (+1.3) at the pH (7.2) of the CCF; however it only led to limited adsorption of the BsAb1 product on the negatively charged elements of the X0HC filter media [ 21 ] at the relatively high salt concentration. Therefore, electrostatic interactions likely played very little role in retaining the a-LC variant due to its negligible positive charge.…”

“…The properties of the Millistak+® D0HC and X0HC filters have been recently investigated using confocal microscopy [ 21 ]. It was shown that the X0HC filter is composed of two layers both containing mainly naturally derived diatomaceous earth (DE) filter aids and a small portion of cellulosic fibers.…”

“…The larger pore size of the D0HC filter also potentially leads to less proximity of binding sites which reduces the chance of multiple interactions between the bound species and the filter media. In addition, the adsorptive properties of these two filters can be varied due to the potential differences in the type and size of the DE particles and cellulose fibers used between these two filters which were neither discussed by literature [ 21 ] nor provided by the manufacturer. The naturally derived DE component contains porous particles that are mainly composed of silicon (Si) and can differ in shape, size, morphology, and porosity.…”

“…The product-related impurities were mainly reduced by the X0HC filter with minimal retention on the D0HC filter observed. The distinct adsorptive properties of the filters were likely due to their varied binding surface area and binding sites proximity, caused by different composition or potentially different type and size of their structural elements (i.e., silica of DE filter aids as the dominant element in the X0HC filter compared with cellulosic fibers as the major component in the D0HC filter [ 21 ]). The LMW variants preferentially retained on the X0HC filter were identified to include one of the light chains present in the structure of the BsAb1 molecule (a-LC), its dimer (a-LC dimer), and a homodimer.…”

Comparison of host cell protein removal by depth filters with diatomaceous earth and synthetic silica filter aids using model proteins

“…The X0SP also showed improved HCP removal for several CHO‐derived antibodies. Parau et al (2022) found higher throughput during initial clarification of CHO cell culture fluid using the X0SP, with the X0SP and X0HC showing similar DNA breakthrough. Confocal microscopy was used to identify the location of foulants within the depth filters, with the greater capacity of the X0SP arising from better utilization of the second layer of the X0SP media.…”