Chromatographic capture of cells to achieve single stage clarification in recombinant protein purification

Abstract

Recent advancements in cell culture engineering have allowed drug manufacturers to achieve higher productivity by driving higher product titers through cell line engineering and high‐cell densities. However, these advancements have shifted the burden to clarification and downstream processing where the difficulties now revolve around removing higher levels of process‐ and product‐related impurities. As a result, a lot of research efforts have turned to developing new approaches and technologies or process optimization to still deliver high quality biological products while controlling cost of goods. Here, we explored the impact of a novel single use technology employing chromatographic principle‐based clarification for a process‐intensified cell line technology. In this study, a 16% economic benefit ($/g) was observed using a single‐use chromatographic clarification compared to traditional single‐use clarification technology by improving the overall product cost through decreased operational complexity, higher loading capacity, increased product recovery, and higher impurity clearance. In the end, the described novel chromatographic approach significantly simplified and enhanced the cell culture fluid harvest unit operation by combining the reduction of insoluble and key soluble contaminants of the harvest fluid into a single stage.

Single‐use centrifugal separator enables intensification of the clarification process in biomanufacturing of recombinant proteins

“…Figure 2(F) (see colored curves) shows the particle size distribution measured by DLS of the light phase obtained from the centrifuge for all nine operating conditions. The particle size distribution ‘generally’ overlaps for all the conditions and consists of a target protein peak of ~10 nm and a broad peak of ~200 nm indicative of various soluble and insoluble contaminants such as cell debris and rDNA (residual DNA); 32,36‐38 the presence of these contaminants ≤1 μm in size necessitates subsequent secondary‐stage clarification as discussed in the later sections. Further inspection of data shown in Fig.…”

“…While X0SP contains a synthetic depth filter media consisting of polyacrylic fibers and silica, A1HC, X0HC, and F0HC contain depth filter media made with naturally derived components, including cellulose and diatomaceous earth 18,19,46 . These filters combine size‐based retention and multiple weak adsorption interactions (based mainly on charge and hydrophobic interactions) to capture a range of soluble and insoluble contaminants, an inherently different mechanism compared to 3M Emphaze, where the capture of contaminants is based entirely on charge‐based interactions 18,19,32,33 . Figure 4(A) shows the throughput data in L m −2 obtained from various filters (data over the light green background) obtained by passing the light phase at 150 LMH until the differential pressures across the filters reached ≥15 psi (see Fig.…”

“…[29][30][31] Figure 1 (C) shows these heightened costs associated with clarifying a 2000-L cGMP (current good manufacturing practice) batch using depth filters over the last 2 years as a result of increased cell culture densities compared to the previous years. Despite recent improvements in depth-filter-based clarification schemes, such as bioreactor feed acidification or pretreatment with flocculants and the design of chromatographic filters with strong adsorption capabilities (such as 3M Harvest RC), 15,18,21,32,33 implementation of depth filters for clarification of high cell-density cultures remains a challenge.…”

Leveraging mathematical models for optimizing filter utility at manufacturing scale

“…In some cases these models are sufficient to describe the fouling processes observed during filtration (Goldrick et al, 2017; Sampath et al, 2014). However, complex biological material consists of an assortment of cellular debris with a range of charges and sizes (Almeida et al, 2022). In cases where the classic filter fouling models do not capture the fouling process, combination models incorporating two or more of the classic filter fouling mechanisms have been shown to describe the fouling behavior of complex biological material (Bolton et al, 2006).…”

“…However, complex biological material consists of an assortment of cellular debris with a range of charges and sizes (Almeida et al, 2022).…”