Naveenkumar Singh, Ph.D.

A single-stage clarification was developed using a single-use chromatographic clarification device (CCD) to recover a recombinant protein from Chinese Hamster Ovary (CHO) harvest cell culture fluid (HCCF). Clarification of a CHO HCCF is a complex and costly process, involving multiple stages of centrifugation and/or depth filtration to remove cells and debris and to reduce process-related impurities such as host cell protein (HCP), nucleic acids, and lipids. When using depth filtration, the… Show more

A single-stage clarification was developed using a single-use chromatographic clarification device (CCD) to recover a recombinant protein from Chinese Hamster Ovary (CHO) harvest cell culture fluid (HCCF). Clarification of a CHO HCCF is a complex and costly process, involving multiple stages of centrifugation and/or depth filtration to remove cells and debris and to reduce process-related impurities such as host cell protein (HCP), nucleic acids, and lipids. When using depth filtration, the filter train consists of multiple filters of varying ratios, layers, pore sizes, and adsorptive properties. The depth filters, in combination with a 0.2-micron membrane filter, clarify the HCCF based on size-exclusion, adsorptive, and charge-based mechanisms, and provide robust bioburden control. Each stage of the clarification process requires time, labor, and utilities, with product loss at each step. Here, use of the 3M™ Harvest RC Chromatographic Clarifier, a single-stage CCD, is identified as an alternative strategy to a three-stage filtration train. The CCD results in less overall filter area, less volume for flushing, and higher yield. Using bioprocess cost modeling, the single-stage clarification process was compared to a three-stage filtration process. By compressing the CHO HCCF clarification to a single chromatographic stage, the overall cost of the clarification process was reduced by 17%–30%, depending on bioreactor scale. The main drivers for the cost reduction were reduced total filtration area, labor, time, and utilities. The benefits of the single-stage harvest process extended throughout the downstream process, resulting in a 25% relative increase in cumulative yield with comparable impurity clearance. Show less

This tutorial describes the use of novel disposable flow path chromatography systems that can be used for the rapid, inexpensive purification of products for gene therapy (viral vectors). These benchtop systems employ lower flow rate ranges, have smaller holdup volumes and smaller footprints, and are suitable for use with the smaller bioreactors used in gene therapy.

BACKGROUND

‘Expanded’ composite materials are of interest as an alternative, or as a supplement, to packed‐bed chromatography during bioproduct recovery and purification. Functionalized non‐woven fabrics and mega‐porous bodies are examples of systems that showed promise. However, there is scarce information on their suitability to capture and release plasmid DNA (pDNA), an important type of product employed in gene therapy.

RESULTS

Composite adsorbents were prepared using… Show more

BACKGROUND

‘Expanded’ composite materials are of interest as an alternative, or as a supplement, to packed‐bed chromatography during bioproduct recovery and purification. Functionalized non‐woven fabrics and mega‐porous bodies are examples of systems that showed promise. However, there is scarce information on their suitability to capture and release plasmid DNA (pDNA), an important type of product employed in gene therapy.

RESULTS

Composite adsorbents were prepared using either chemical (CG‐DEAE‐NW) or gamma‐irradiated graft‐polymerization (GIR‐DEAE‐MP), and subsequently modified to have diethylamino ethanol (DEAE) functionality. Capture experiments showed that pDNA can actually reversibly bind to the two mentioned adsorbents, with capacity values of 2.4 and 1.3 mg per mL, respectively. These values are in the range of what can be expected from commercial beaded adsorbents but lower that the values expected from monoliths.

CONCLUSIONS

Expanded materials, due to their high voidage, may present limited capacity for pDNA. However, such materials are able to bind proteins and other contaminants from bacterial lysate, opening the way for their utilization in the ‘negative’ mode. Show less

Fibrous adsorbents were subjected either to chemical treatment or gamma irradiation to introduce epoxide groups onto their cellulosic backbone using glycidyl methacrylate. These epoxide moieties were modified to have diethylaminoethanol (DEAE) as well as quaternary ammonium (Q) functionalities. The resulting anion exchange adsorbents were characterized by their FTIR spectra and ionic capacities. The fiber-based adsorbent systems showed similar packing efficiency to the commercially available… Show more

Fibrous adsorbents were subjected either to chemical treatment or gamma irradiation to introduce epoxide groups onto their cellulosic backbone using glycidyl methacrylate. These epoxide moieties were modified to have diethylaminoethanol (DEAE) as well as quaternary ammonium (Q) functionalities. The resulting anion exchange adsorbents were characterized by their FTIR spectra and ionic capacities. The fiber-based adsorbent systems showed similar packing efficiency to the commercially available adsorbents, where the Péclet number values were ⩾60, suggesting near-plug-flow conditions. The total ionic capacities obtained for these chemically grafted adsorbents were ca. 400 mmol/L. These adsorbents showed dynamic binding capacities (DBC) of ca. 48 mg/mL for bovine serum albumin (BSA). Protein binding capacities obtained from chemical grafting initiation techniques were 18-fold-higher than radiation-induced techniques. The advantage of these adsorbents lies in their high operational flow rates while maintaining their high binding capacities.

Keywords: Column packing; Epoxide functionalization; Protein purification; Anion-exchange chromatography; Cellulosic adsorbents; Radiation-induced grafting; Fiber-based adsorbents. Show less

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90 %. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a… Show more

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90 %. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

Keywords: Protein purification; Graft-copolymerization; Megaporous cryogels; IMAC Affinity chromatography. Show less

Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27 ± 3 mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a… Show more

Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27 ± 3 mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119 mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10–100 μm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties. Show less

Megaporous cryogels with metal-ion affinity functionality, which possess enhanced protein-binding ability, were synthesized and their properties were investigated. These highly porous materials (pore sizes up to 100 μm) allowed the direct capture of a recombinant His6-tagged protein from a partially clarified extract. The total ligand density of the material was found to be 770 μmol/g. Application of a partially clarified cell extract in order to recover a His6-tagged protein (NAD(P)H-dependent… Show more

Megaporous cryogels with metal-ion affinity functionality, which possess enhanced protein-binding ability, were synthesized and their properties were investigated. These highly porous materials (pore sizes up to 100 μm) allowed the direct capture of a recombinant His6-tagged protein from a partially clarified extract. The total ligand density of the material was found to be 770 μmol/g. Application of a partially clarified cell extract in order to recover a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) yielded 12 mg of highly purified recombinant product per gram of adsorbent. Increased dynamic binding capacities were observed upon larger degrees of grafting, although some reduction in the quality of the system hydrodynamics was also observed. Nevertheless, these immobilized metal-ion affinity cryogels show potential for a convenient single-step purification of recombinant proteins from raw cell extracts without the need for laborious pre-chromatographic sample clean-up procedures. Show less