Unclarified Broth Research Articles

A quaternary amine cryogel column for chromatographic capture of l-asparaginase

L-asparaginase is a therapeutic enzyme used in therapies of acute lymphoblastic leukemia. Its purification conventionally uses several unit operations, and usual protocols lead to low yields and extensive processing times, which reduce productivity and increases process costs. In this study, we produced a supermacroporous cryogel activated with polyethyleneimine, followed by quaternization with epichlorohydrin resulting in a quaternary ammonium anion-exchanger. The anionic cryogel had a ligand density of 121.6 mmol/L and the HETP ranged from 0.18 to 0.27 cm. The activated cryogel was applied to capture L-asparaginase from filtered broth. Yield and purification fold (PF) were optimized using the response-surface methodology, resulting in optimum values of 56 % and 7.6, respectively. The effect of flow velocity over PF was also analyzed, and the results showed that PF had an average value of 8.5 and did not vary with the fluid velocity, confirming the convective mass transfer property of the activated cryogel. The results showed that the activated cryogel shows advantages in using unclarified broth without pretreatment and works at a high flow rate with the perspective of reducing unit production cost.

Vortex flow reactor assessment for the purification of monoclonal antibodies from unclarified broths

The vortex flow reactor (VFR) can be used in many chemical engineering applications. This paper assesses its novel use in the purification of monoclonal antibodies from cell broth. To this end, the IgG2a antibody was purified from the unclarified fermentation broth of transgenic mouse 55/6 hybridoma cells. Visual experiments showed that the VFR worked in the laminar vortices flow regime and the vortices displaced slightly faster than the axial flow. The VFR has the advantage of creating two sorts of flows: axial flow to produce the expanded bed and an extra vortex flow to avoid channeling and stabilize the expanded bed, the hydrodynamic behavior of which is plug flow with an experimental Pèclet number higher than 20. The pH was adjusted in the untreated fermentation broth, which was directly introduced into the reactor thus reducing the number of stages. The IgG2a purification was carried out in a single device via two steps: antibody adsorption in the expanded bed and antibody elution in the settled bed using Streamline rProtein A. A thirty-fold increase in the high-purity antibody concentration was achieved at the top of the pH5 elution peak with a total recovery of 93.1% (w/w) between elution peaks pH 5 and 3.

Single-step purification of chitosanases from Bacillus cereus using expanded bed chromatography

A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01. The purification and characterization of two chitosanases were studied. The purification assay was accomplished by ion exchange expanded-bed chromatography. Experiments were carried out in the presence and in the absence of cells through different expansion degree to evaluate the process performance. The adsorption experiments demonstrated that the biomass does not affect substantially the adsorption capacity of the matrix. The enzyme bound to the resin with the same extent using clarified and unclarified broth (0.32 and 0.30U/g adsorbent, respectively). The fraction recovered exhibited 31% of the yield with a 1.26-fold increase on the specific activity concerned to the initial broth. Two chitosanases from different elution steps were recovery. Chit A and Chit B were stable at 30–60°C, pH 5.5–8.0 and 5.5–7.5, respectively. The highest activity was found at 55°C, pH 5.5 to Chit A and 50°C, pH 6.5 to Chit B. The ions Cu2+, Fe2+ and Zn2+ indicated inhibitory effect on chitosanases activities that were significantly activated by Mn2+. The methodology applied in this study enables the partial purification of a stable chitosanase using a feedstock without any pre-treatment using a single-step purification.

Expanded-bed adsorption immobilized-metal affinity chromatography

The protocol describes a method for capture of secreted hexahistidine-tagged proteins using expanded-bed adsorption immobilized-metal affinity chromatography. The starting material for the procedure is any crude feedstock that contains a histidine (His)-tagged target protein. The protocol is exemplified using unclarified broth from Pichia pastoris fermentation as feedstock. The protocol can be used for laboratory studies or as part of a process for production of recombinant biotherapeutics to standards of good manufacturing practice. It takes approximately 5 h to purify proteins from 10 liters of feedstock and a further 5-6 h to sterilize and regenerate the column.

Fluidized bed adsorption as a primary recovery step in protein purification.

Fluidized bed adsorption has been introduced as an integrative technology combining clarification, concentration, and initial purification in a single step. In the paper presented here, the use of fluidized adsorbents in the primary recovery of proteins starting from unclarified broths is reviewed. First the principle of fluidizing adsorbent particles is discussed, subsequently possible experimental procedures for whole broth adsorption are demonstrated. The system parameters governing the performance of the sorption process in a fluidized bed are discussed in the second part of the paper and considerations on how operating parameters and process design influence the limiting steps are provided. Finally, examples for the successful operation of whole broth adsorption employing fluidized adsorbents are shown and conditions are defined under which this technology may be an alternative to traditional protein purification methods.

Bioproduct Recovery From Unclarified Broths and Homogenates Using Immobilized Adsorbents

AbstractThe use of reversibly “immobilized” (gel‐matrix entrapped or gel‐membrane encapsulated) adsorbents for the recovery of protein products from unclarified broths or cell homogenates containing difficult to remove solids was investigated. The hydrogel membrane or matrix acts as a protective non‐fouling barrier that allows the diffusion of the desired bioproduct, but it excludes colloidal solids such as whole cells, cell debris, and macromolecular precipitates. This feature not only prevents the fouling of the adsorbent, it also enhances product separation. The concept of kinetic selectivity, which depends on the differences in the diffusion rates of various adsorbing species is also introduced. The utility of hydrogel matrix or membrane in enhancing the kinetic selectivity of smaller proteins from a crude mixture was also investigated using model systems.