Advances in Primary Recovery: Centrifugation and Membrane Technology

Abstract

Significant and continual improvements in upstream processing for biologics have resulted in challenges for downstream processing, both primary recovery and purification ( 1). Given the high cell densities achievable in both microbial and mammalian cell culture processes, primary recovery can be a significant bottleneck in both clinical and commercial manufacturing. The combination of increased product titer and low viability leads to significant relative increases in the levels of process impurities such as lipids, intracellular proteins and nucleic acid versus the product. In addition, cell culture media components such as soy and yeast hydrolysates have been widely applied to achieve the cell culture densities needed for higher titers ( 2, 3). Many of the process impurities can be negatively charged at harvest pH and can form colloids during the cell culture and harvest processes. The wide size distribution of these particles and the potential for additional particles to be generated by shear forces within a centrifuge may result in insufficient clarification to prevent fouling of subsequent filters. The other residual process impurities can lead to precipitation and increased turbidity during processing and even interference with the performance of the capturing chromatographic step. Primary recovery also poses significant challenges owing to the necessity to execute in an expedient manner to minimize both product degradation and bioburden concerns. Both microfiltration and centrifugation coupled with depth filtration have been employed successfully as primary recovery processing steps. Advances in the design and application of membrane technology for microfiltration and dead‐end filtration have contributed to significant improvements in process performance and integration, in some cases allowing for a combination of multiple unit operations in a given step. Although these advances have increased productivity and reliability, the net result is that optimization of primary recovery processes has become substantially more complicated. Ironically, the application of classical chemical engineering approaches to overcome issues in primary recovery and purification (e.g., turbidity and trace impurity removal) are just recently gaining attention ( 4). Some of these techniques (e.g., membrane cascades, pretreatment, precipitation, and the use of affinity tags) are now seen almost as disruptive technologies ( 5). This paper will review the current and potential future state of research on primary recovery, including relevant papers presented at the 234th American Chemical Society (ACS) National Meeting in Boston.

Technology advancements in antibody purification

“…Centrifugation facilitates separation of the antibody-containing supernatant from the cells and cellular debris; however, parameters such as g-force, residence time, and discharge frequency can influence clarification efficiency and can vary greatly when moving from small-scale to large-scale centrifugation. 35 Membrane filtration, such as microfiltration, ultrafiltration, and depth filtration allow separation of the small molecules by size, while charged ultrafiltration can separate proteins by both charge and size. For example, cellulose-based ultrafiltration membranes have been used to separate different therapeutic mAbs from CHO cells.…”

“…For example, cellulose-based ultrafiltration membranes have been used to separate different therapeutic mAbs from CHO cells. 1,35,36 Microfiltration membrane chromatography removes biological entities (DNA, viruses, and protein contaminants) from fluids using a microporous membrane filter, examples of which include polypropylene or polyester. The membranes are stacked ten to 15 layers deep and are packed into a small disposable cartridge.…”

“…Particle retention is thought to occur by a combination of size exclusion and adsorption through hydrophobic, ionic, and other interactions. 35 Chemical methods for the alleviation of turbidity include flocculation, precipitation, and crystallization. Flocculation can be used to remove particulate matter of submicrometer size.…”

“…It can be achieved using 30 mM calcium chloride followed by 20 mM potassium phosphate (the calcium phosphate forms a precipitate around the cellular debris). 1,35 Precipitation is an effective technique used to remove antibody from its sample matrix. Antibody precipitation can be achieved using ammonium sulfate, octonic acid, or polyethylene glycol (PEG).…”

Purification of recombinant baculoviruses for gene therapy using membrane processes

“…Moreover, the selected pore size allowed for a more effective utilization of the entire depth of the porous filter for rBVs. 17 Concentration of rBVs and removal of low molecular weight contaminants was performed using tangential flow filtration (TFF); this is a technique routinely used at large scale to concentrate and purify viral products. 14 The best retentate flow rates and transmembrane pressure (TMP) operating conditions were determined aiming at an optimized utilization of the membrane while reducing membrane fouling and consequent product loss (Figure 1a).…”