Stability of expanded beds during the application of crude feedstock

Expanded bed adsorption is an integrated technology that allows the introduction of a particle containing feedstock without the risk of blocking the bed. Provided a perfectly classified fluidized bed (termed expanded bed) is formed in the crude feed, a sorption performance comparable to packed beds is found. During the application of biomass containing samples to stable expanded beds an increase in bed expansion due to the higher density and viscosity of the feed is encountered. In this article it is investigated whether the expanded bed condition is also fulfilled during the transition in bed expansion from lower to higher density (i.e., from an equilibration buffer to a biomass containing feedstock). Residence time distribution analyses were performed by using model systems and a yeast suspension during this transition phase. It is shown that in systems in which the biomass does not interact with the fluidized stationary phase, the perfectly classified fluidization is maintained also during this transition phase regardless of the type of feedstock. Additional bed expansion takes place in an "ordered" manner without compromising bed stability. In case of biomass/adsorbent interactions, a deterioration in bed stability is found directly when the crude feed is loaded.     

Minimising biomass/adsorbent interactions in expanded bed adsorption processes: a methodological design approach

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from crude feedstock. Interactions between solid matter in the feed suspension and fluidised adsorbent particles influence bed stability and therefore have a significant impact on protein adsorption in expanded beds. In order to design efficient and reliable EBA processes a strategy is needed, which allows to find operating conditions, where these adverse events do not take place. In this paper a methodological approach is presented, which allows systematic characterisation and minimisation of cell/adsorbent interactions with as little experimental effort as possible. Adsorption of BSA to the anion exchanger Streamline Q XL from a suspension containing S. cerevisiae cells was chosen as a model system with a strong affinity of the biomass towards the stationary phase. Finite bath biomass adsorption experiments were developed as an initial screening method to estimate a potential interference. The adhesiveness of S. cerevisiae to the anion exchanger could be reduced significantly by increasing the conductivity of the feedstock. A biomass pulse response method was used to find optimal operation conditions showing no cell/adsorbent interactions. A good correlation was found between the finite bath test and the pulse experiment for a variety of suspensions (intact yeast cells, E. coli homogenate and hybridoma cells) and adsorbents (Streamline Q XL, DEAE and SP), which allows to predict cell/adsorbent interactions in expanded beds just from finite bath adsorption tests. Under the optimised operating conditions obtained using the prior methods, the stability of the expanded bed was investigated during fluidisation in biomass containing feedstock (up to 15% yeast on wet weight basis) employing residence time distribution analysis and evaluation by an advanced model. Based on these studies threshold values were defined for the individual experiments, which have to be achieved in order to obtain an efficient EBA process. Breakthrough experiments were conducted to characterise the efficiency of BSA adsorption from S. cerevisiae suspensions in EBA mode under varying operating conditions. This allowed to correlate the stability of the expanded bed with its sorption efficiency and therefore could be used to verify the threshold values defined. The approach presented in this work provides a fast and simple way to minimise cell/adsorbent interactions and to define a window of operation for protein purification using EBA.     

Effect of adsorbent porosity on performance of expanded bed chromatography of proteins

Expanded bed or fluidized bed adsorption has emerged as an important unit operation in downstream processing of proteins. A number of specifically designed commercial adsorbents are available today for expanded bed purification of proteins. Protein purification essentially requires adsorbent matrices that have large pore size. Very large pore size or macroporous adsorbents can provide high efficiency in packed beds even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the macropores. This is reflected in leveling off of HETP (height equivalent to theoretical plate) versus flow curve after a threshold velocity. Expanded bed operation, on the other hand, can also show plateauing of the HETP curve, but not necessarily on account of macroporosity of adsorbent. It is shown in this article how any adsorbent intended for protein adsorption in expanded bed mode can give plateauing HETP curve, regardless of pore size. As a result, RTD measurements on an expanded bed can give equal, and at times better, performance than a corresponding packed bed. Large pore size, on the other hand, can result in lesser retention of biomass and easy flushing of the adsorbent to obtain an entirely particulate-free adsorbent prior to the product elution step. Adsorbent with larger pores is also shown to provide faster and more efficient elution both in packed and expanded bed modes.     

The influence of complex biological feedstock on the fluidization and bed stability in expanded bed adsorption

The stability of expanded bed adsorption systems (EBA) was studied in biomass containing culture broth by residence time distribution (RTD) experiments, using pulse inputs of fluorescent molecules as tracers. Different commercial adsorbents (Streamline DEAE, SP, Phenyl, Chelating, and AC) were tested at various biomass concentrations (2.5–12 %, wet weight) of whole (Saccharomyces cerevisiae) yeast, yeast cell homogenate, and Escherichia coli homogenate. Analyzing the RTD according to the PDE model (PDE: axially dispersed plug‐flow exchanging mass with stagnant zones) allowed the calculation of three parameters: the number of transfer units for mass exchange between mobile and stagnant fraction (N), the Peclet number for overall axial dispersion (P), and the mobile fraction of the liquid in axially dispersed plug flow (φ). When fluidization was performed in particle‐free buffer the normalized response signal (after perfect input pulse) was symmetric (N:0; P: 50–100; φ: 1), thus, demonstrating the formation of a homogeneous fluidized (expanded) bed. Upon application of suspended biomass the RTD was skewed, depending on the adsorbent used and the type and level of biomass present in the sample. This situation leads to three different characteristic pictures: the well‐fluidized system (N: ≥ 7–10; P: ≥ 40; φ: 0.80–0.90), the system exhibiting bottom channeling (N: < 1–2; P: ≥ 40; φ: 0.5–0.7) and, the system where extensive agglomeration develops (N: 4–7; P: 20–40; φ: < 0.5). These results demonstrate that changes in the hydrodynamics of EBA already take place in the presence of moderate concentrations of biomass. Furthermore, those changes can be quantitatively described mainly in terms of the fraction of stagnant zones in the system, which are formed due to the interaction of biomass and adsorbent. The technique described here can be used to evaluate a certain combination of adsorbent and biomass with regard to its suitability for expanded bed adsorption from whole broth. © 1999 John Wiley & Sons, Inc. Biotechol Bioeng 64: 484–496, 1999.     

Separation of nattokinase fromBacillus subtilis fermentation broth by expanded bed adsorption with mixed-mode adsorbent

Mixed-mode hydrophobic/ionic matrices exhibit a salt-tolerant property for adsorbing target protein from high-ionic strength feedstock, which allows the application of undiluted feedstockvia an expanded bed process. In the present work, a new type of mixed-mode adsorbent designed for expanded bed adsorption, Fastline PRO^®, was challenged for the capture of nattokinase from the high ionic fermentation broth ofBacillus subtilis. Two important factors, pH and ion concentration, were investigated with regard to the performance of nattokinase adsorption. Under initial fermentation broth conditions (pH 6.6 and conductivity of 10 mS/cm) the adsorption capacity of nattokinase with Fastline PRO was high, with a maximum capacity of 5,350 U/mL adsorbent. The elution behaviors were investigated using packed bed adsorption experiments, which demonstrated that the effective desorption of nattokinase could be achieved by effecting a pH of 9.5. The biomass pulse response experiments were carried out in order to evaluate the biomass/adsorbent interactions betweenBacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containingBacillus subtilis cells. Finally, an EBA process, utilizing mixed-mode Fastline PRO adsorbent, was optimized to capture nattokinase directly from the fermentation broth. The purification factor reached 12.3, thereby demonstrating the advantages of the mixed-mode EBA in enzyme separation.     

Target control of cell disruption to minimize the biomass electrostatic adhesion during anion-exchange expanded bed adsorption

Expanded bed adsorption (EBA) is an integrative unit operation for the primary recovery of bioproducts from crude feedstock. Biomass electrostatic adhesion often leads to bad bed stability and low adsorption capacity. The results indicate that effective cell disruption is a potential approach to reduce the biomass adhesion during anion-exchange EBA. Two common cell disruption methods (sonication treatment and high-pressure disruption with a French press) were investigated in the present work. The mean size of cell debris reduced dramatically during the cell disruption process, and the absolute value of the zeta potential of cell debris also decreased significantly as the mean size reduced. The biomass transmission index (BTI) obtained through the biomass pulse response experiment was used to quantitatively evaluate the biomass-adsorbent interaction. Combining the influences of zeta potential of adsorbent (zetaA), zeta potential of biomass (zetaB), and biomass mean size (dB), the parameter of (-zetaA.zetaB.dB) was explored as a reasonable indicator of biomass adhesion in expanded beds. A good linear correlation was confirmed between BTI and (-zetaA.zetaB.dB) for all biomass and cell disruption conditions tested, which was independent of the cell disruption methods. A target parameter (-zetaA.zetaB.dB) of 120 mV2mum was derived for BTI above 0.9, which meant a very slight influence of biomass on the stability of the expanded bed. This criterion could be used as a rational control target for cell disruption processes in EBA applications.     

Optimization Considerations for the Purification of α1-Antitrypsin Using Silica-Based Ion-Exchange Adsorbents in Packed and Expanded Beds

The influences of the fluid superficial velocity, sample concentration, loading volume, and wash cycle on the recovery and corresponding purification factors for α₁-antitrypsin [syn. α₁-proteinase inhibitor (α₁-PI) ] from crude mixtures of human plasma proteins were investigated using packed and expanded beds of DEAE-Spherodex LS. As part of this study, the effect of fluid superficial velocity on the bed dispersion number (D _v) and dispersion coefficient (D) for this adsorbent in expanded beds was determined with feedstocks containing human serum albumin (HSA), the most abundant of the contaminating proteins in human plasma protein preparations used for the isolation of α₁-PI. When multicomponent protein feedstocks prepared from human plasma were examined with DEAE-Spherodex LS, reduced chromatographic productivity was observed for α₁-PI as the extent of column utilization and the superficial velocity were increased, yet the opposite trend was evident for HSA. In particular, higher adsorption capacities and recoveries were obtained for α₁-PI at lower fluid superficial velocities with both packed and expanded bed conditions. These findings indicate that for process scale purifications of α₁-PI from multicomponent feedstocks with expanded beds containing this silica-based ion-exchange adsorbent, the optimal range of superficial velocities to achieve the highest bed productivity will not be synonymous with maximally fluidized modes of operation. Rather, the results confirm that the adsorbent has an optimum operational performance when fluidization procedures corresponding to plug flow expansion are employed for the capture of α₁-PI. These findings also indicate that advantage can be taken of displacement effects between closely related protein species with packed and expanded bed systems containing the DEAE-Spherodex LS type of ion-exchange porous silicas.     

Expanded bed adsorption at production scale: Scale-up verification, process example and sanitization of column and adsorbent

Expanded bed adsorption is a technique for recovery of biomolecules directly from unclarified feedstocks. The work described here demonstrates that expanded bed adsorption is a scaleable technique. The methods used to test scaleability were “determination of degree of bed expansion”, “determination of axial dispersion” and “determination of protein breakthrough capacity”. The performance of a production scale expanded bed column with 600?mm diameter was tested using these methods and the results were found to be consistent with the results obtained from lab scale and pilot scale expanded bed columns. The scaleability and function of the expanded bed technique was also tested by performing a “process example”: a purification mimicking a real process using a yeast culture spiked with bovine serum albumin as feedstock. The results show that the 600?mm diameter production scale column was as efficient as a 25?mm diameter lab scale column in recovering bovine serum albumin from the unclarified yeast culture. The production scale runs were fully automated using a software controlled system containing an adaptor position sensor and an adsorbent sensor. A cleaning study was performed which showed that after use of a proper cleaning protocol, no surviving microorganisms could be detected in the column or in the adsorbent.     

Purification of recombinant nucleocapsid protein of Newcastle disease virus from unclarified feedstock using expanded bed adsorption chromatography

In the present work, a single-step purification of recombinant nucleocapsid protein (NP) of the Newcastle disease virus (NDV) directly from unclarified feedstock using an expanded bed adsorption chromatography (EBAC) was developed. Streamline 25 column (ID = 25 mm) was used as a contactor and Streamline chelating adsorbent immobilized with Ni2+ ion was used as affinity adsorbent. The dynamic binding capacity of Ni2+ -loaded Streamline chelating adsorbent for the NP protein in unclarified feedstock was found to be 2.94 mg ml(-1) adsorbent at a superficial velocity of 200 cm h(-1). The direct purification of NP protein from unclarified feedstock using expanded bed adsorption has resulted in a 31% adsorption and 9.6% recovery of NP protein. The purity of the NP protein recovered was about 70% and the volume of processing fluid was reduced by a factor of 10. The results of the present study show that the IMA-EBAC developed could be used to combine the clarification, concentration and initial purification steps into a single-step operation.     

Influence of biomass accumulation on bed expansion characteristics of a down-flow anaerobic fluidized-bed reactor

This article describes the bed expansion characteristics of a down-flow anaerobic fluidized bed reactor treating a synthetic wastewater. Experiments were carried out in a 0.08 m diameter and 1 m length PVC column. The carrier used was ground perlite (an expanded volcanic rock). Particles characteristics were 0.968 mm in diameter, specific density of 213 kg x m-3 and Umf (minimal fluidization velocity): 2.3 m x h-1. Experimental data of terminal velocities and bed expansion parameters at several biofilm thicknesses were compared to different models predicting the bed expansion of up-flow and down-flow fluidized beds. Measured bed porosities at different liquid superficial velocities for the different biofilm thicknesses were in agreement with the Richardson-Zaki model, when Ut (particle terminal velocity) and n (expansion coefficient) were calculated by linear regression of the experimental data. Terminal velocities of particles at different biofilm thicknesses calculated from experimental bed expansion data, were found to be much smaller than those obtained when Cd (drag coefficient) is determined from the standard drag curve (Lapple and Sheperd, 1940) or with others' correlations (Karamanev and Nikolov, 1992a,b). This difference could be explained by the fact that free-rising particles do not obey Newton's law for free-settling, as proposed by Karamanev and Nikolov (1992a,b) and Karamanev et al. (1996). In the present study, the same free-rising behavior was observed for all particles (densities between 213 and 490 kg x m-3).     

Zeta potential as a diagnostic tool to evaluate the biomass electrostatic adhesion during ion-exchange expanded bed application

Expanded bed adsorption is an integrative technology in downstream processing allowing the direct capture of target proteins from biomass (cells or cell debris) containing feedstocks. Potential adhesion of biomass on the surface of adsorbent, however, may hamper the application of this technique. Since the electrostatic forces dominate the interactions between biomass and adsorbent, the concept of zeta potential was introduced to characterize the biomass/adsorbent electrostatic interactions during expanded bed application. The criterion of zeta potential evaluation proposed in the previous paper (Biotechnol Bioeng, 83(2):149-157, 2003) was verified further with the experimental validation. The zeta potential of intact cells and homogenates of four microorganisms (Escherichia coli, Bacillus subtilis, Pichia pastoris, and S. cerevisiae) were measured under varying pH and salt concentration, and two ion-exchange adsorbents (Streamline DEAE and Streamline QXL) were investigated. The biomass transmission index (BTI) from the biomass pulse response experiments was used as the indicator of biomass adhesion in expanded bed. Combining the influences from zeta potential of adsorbent (zeta(a)), zeta potential of biomass (zeta(b)) and biomass size (d), a good relationship was established between the zeta potential parameter (-zeta(a)zeta(b)d) and BTI for all experimental conditions. The threshold value of parameter (-zeta(a)zeta(b)d) can be defined as 120 mV2 microm for BTI above 0.9. This means that the systems with (-zeta(a)zeta(b)d) < 120 show neglectable electrostatic bio-adhesion, and would have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions.     

Development of operating conditions for protein purification using expanded bed techniques: the effect of the degree of bed expansion on adsorption performance

A strategy for the optimization of an expanded bed adsorption process has been developed by studying a model system involving the adsorption of lysozyme onto the adsorbent STREAMLINE SP. The hydrodynamic and adsorption properties of this ion exchange adsorbent in a variety of viscosities of feedstocks have been compared by analyzing bed expansion characteristics, liquid phase dispersion characteristics, equilibrium adsorption isotherms, and mass transfer characteristics. Additionally, the influences of the degree of bed expansion on adsorption performance have been investigated by frontal analysis. In these experiments, viscous feedstocks were simulated by the inclusion of glycerol in the adsorption buffers. Breakthrough curves for lysozyme were characterized and compared in terms of overall purification processing time and productivity. On the basis of these results, the relative productivities of different operating modes with the same process liquid were found to be almost the same. However, the processing time for each purification cycle decreased with increasing velocity of process liquid. It is demonstrated that an adsorption process carried out at a constant degree of bed expansion (twice its settled bed height, corresponding to bed voidage of 0.7) is more efficient, when characterized by the apparent dynamic binding capacity, than operation at a constant liquid velocity of 300 cm/h. These results have significant implications on the design and operation of the expanded bed adsorption procedures. The advantages and problems encountered in the use of expanded bed techniques for the direct extraction of proteins from unclarified feedstocks are also discussed. (c) 1996 John Wiley & Sons, Inc.     

Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption.

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.     

A study of the influence of yeast cell debris on protein and alpha-glucosidase adsorption at various zones within the expanded bed using in-bed sampling

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.     

Dye–ligand expanded bed adsorption of G6PDH from highly dense unclarified yeast extract

The application of dye–ligand expanded bed chromatography adsorption (EBA) of glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast extract was undertaken by using a commercially available expanded bed column (20 mm i.d.) and UpFront adsorbent (ρ = 1.5 g/mL) from UpFront Chromatography. The influence of biomass concentration on the adsorption capacity was explored by employing yeast extracts containing various biomass concentrations (5–30%, w/v). It was demonstrated that the biomass concentration had little effect on G6PDH adsorption performance. Feedstock containing 15% (w/v) biomass gave a relatively high recovery yield (>90%) of G6PDH compared to feedstock containing 30% (w/v) biomass, which gave a recovery of 75% G6PDH. Nevertheless, the enzyme specific activity of 7 U mg⁻¹ with a purification factor of 6 was achieved in the feedstock containing biomass concentration of 30% (w/v). The generic applicability of dye–ligand as an affinity tool in expanded bed chromatography is discussed.     

Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. (c) 1995 John Wiley & Sons, Inc.     

Cell/adsorbent interactions in expanded bed adsorption of proteins

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidised resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.     

Effect of different operating modes and biomass concentrations on the recovery of recombinant hepatitis B core antigen from thermal-treated unclarified Escherichia coli feedstock

Expanded bed adsorption chromatography (EBAC) is a single pass operation that has been used as primary capture step in various protein purifications. The most common problem in EBAC is often associated with successful formation of a stable fluidized bed during the absorption stage, which is critically dependent on parameters such as liquid velocity, bed height, particle (adsorbent) size and density as well as design of column and type of flow distributor. In this study, residence time distribution (RTD) test using acetone as non-binding tracer acetone was performed to evaluate liquid dispersion characteristics of the EBAC system. A high B(o) number was obtained indicating the liquid dispersion in the system employed is very minimal and the liquid flow within the bed was close to plug flow, which mimics a packed bed chromatography system. Evaluation on the effect of flow velocities and bed height on the performance of Streamline DEAE using feedstock containing heat-treated crude Escherichia coli homogenate of different biomass concentrations was carried out in this study. The advantages and disadvantages as well as the problems encountered during recovery of HBcAg with aforementioned parameters are also discussed in this paper.