Adsorption-desorption of recombinant hepatitis B surface antigen (r-HBsAg) from P. pastoris on a diatomaceous earth matrix: Optimization of parameters for purification

Recombinant hepatitis B surface antigen (r-HBsAg) produced in yeast is adsorbed on a diatomaceous earth matrix for purification purposes. A pH dependence in the adsorption-elution behavior was found. The capacity of celite (Hyflo Super Cei) for adsorbing r-HBsAg increased with decreasing pH. Nonspecific proteins were also adsorbed, but a low pH dependence was found. Elution from the matrix was performed using a basic pH buffer, in which r-HBsAg is more specifically adsorbed/desorbed than contaminant proteins, permitting the purification of the r-HBsAg. A pH of 4.0 was used for adsorption and pH 8.2 was used for desorption. The described protocol allows a purification factor between three- and fivefold with respect to contaminant proteins and sixfold with respect to contaminant DNA. Finally, the adsorption step was successfully scaled-up for production purposes. (c) 1993 John Wiley & Sons, Inc.     

Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody–polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to <1 µg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody. Biotechnol. Bioeng. 2009;102: 1141–1151. © 2008 Wiley Periodicals, Inc.     

Caprylic acid precipitation method for impurity reduction: An alternative to conventional chromatography for monoclonal antibody purification

We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes. Biotechnol. Bioeng. 2012; 109: 2589–2598. © 2012 Wiley Periodicals, Inc.     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

Comparison of different elution conditions for the immunopurification of recombinant hepatitis B surface antigen

An immunoaffinity chromatographic method was developed using a mAb immunosorbent to purify recombinant hepatitis B surface antigen (r-HBsAg) from yeast. Elution conditions using a mAb-coated ELISA were improved to select the best conditions to purify r-HBsAg. The optimum results in terms of total quantitative recovery were obtained using 20 mM Tris pH 11.6. An increase in the CB.Hep-1 mAb (anti-HBsAg) useful immunosorbents half-life and in its yield per cycle was obtained when alkaline elution conditions were used. Moreover, the basic conditions do not affect either the antigenic characteristics or the purity or the molecular integrity of r-HBsAg.     

Simplified purification of equine chorionic gonadotropin (eCG)––an example of the use of magnetic microsorbents for the isolation of glycoproteins from serum

Classical purification of the glycoprotein equine chorionic gonadotropin (eCG) from serum includes pH fractionation with metaphosphoric acid, two ethanol precipitation steps as well as dialysis followed by fixed-bed chromatography. A simplified process requiring only 1/3 of the solvent and improving the yield from 53 to 65% has been developed. The process comprises an ultra-/diafiltration step after the first ethanol precipitation, directly followed by an adsorption/desorption procedure based on magnetic microadsorbents with N,N-diethyl-ammonium functionalization. The process reaches an overall purification factor of eCG of more than 1800 and an average product activity of 1300 IU_ELISA/mg. After adapting the parameters of the fractionation and the type of magnetic microadsorbents, the new concept is likely to be transferable to other serum proteins.     

Sample preparation strategies for quantitative analysis of catalase in red blood cells by elemental mass spectrometry

A sample preparation strategy for the determination of the Fe-containing enzyme catalase (CAT) by Fe specific monitoring in human erythrocytes has been optimized. For this purpose, the combined use of elemental mass spectrometry (via inductively coupled plasma, ICP-MS), molecular mass spectrometry (via MALDI-TOF) and enzymatic activity measurements has been required. The procedure involved haemoglobin precipitation from cell lysate with a solution of ethanol-chloroform and preconcentration of the supernatant by using a Speed-Vac concentrator. Catalase recoveries of about 88 ± 15% could be measured by monitoring the protein enzymatic activity before and after precipitation. Further fractionation of Fe-containing proteins from the preconcentrated extract was achieved by size exclusion chromatography (Superdex 200) with a mobile phase of ammonium acetate (0.05 M, pH 7.4) coupled to ICP-MS (Fe monitoring) and UV/VIS detection (specific absorption of the heme-group at 408 nm). A second dimensional chromatography of the CAT-positive activity fraction was carried out by anion-exchange chromatography (Mono Q 5/50) using for elution a linear gradient of ammonium acetate (0-0.750 M in 15 min). This second step revealed a single Fe-containing species in the chromatogram and permitted the unambiguous characterization of the CAT in such fractions by MALDI-TOF. Column recoveries were evaluated and were quantitative, in terms of Fe bound to protein and CAT activity.     

Introduction of Additional Charges as an Aid in Protein Purification: Isolation of Elongation Factor 2 from Sulfolobus acidocaldarius by Preparative Isoelectric Focusing Before and After ADP-Ribosylation

Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor 2 (EF-2) in eukaryotes and archaebacteria. As the reaction is strictly EF-2 specific and introduces two negative charges into the molecule, the resulting shift in the isoelectric point (pI) by 0.2 pH units was used to establish a new purification method for EF-2 from Sulfolobus acidocaldarius. The cells were lysed with dithiothreitol at pH 9 and EF-2 was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-200, and three isoelectric focusing steps. The EF-2-containing fractions from the first isoelectric focusing step at pH 4-9 were refocused in a more narrow pH-gradient (pH 5-7). The EF-2 peak from the second step was eluted, collecting only the fractions above the pH region where ADP-ribosylated EF-2 would focus. The EF-2 was then ADP-ribosylated with diphtheria toxin and NAD and subjected to further isoelectric focusing (pH 5-7). The EF-2 was almost homogeneous since ADP-ribosylation had shifted it into a region of the pH gradient free of contaminating proteins. Diphtheria toxin was immobilized on CNBr-activated Sepharose to prevent a possible contamination by proteins from the diphtheria toxin preparation which might have the same pI as ADP-ribosylated EF-2. Finally, the ADP-ribosyl group was removed by equilibrium dialysis using diphtheria toxin and nicotinamide at pH 6.3. The obtained EF-2 was active in protein synthesis.     

The effect of morphine tolerance and dependence on cell free protein synthesis

A cell free system consisting of polyribosomes and pH 5 factors of the cytosol was isolated from mouse brain. This system actively promoted the incorporation of radiolabeled amino acids into protein in vitro. Addition of exogenous morphine to a cell free protein synthetic system isolated from chronically morphinized, placebo treated, or naive mouse brain had no effect on the relative synthetic capacity of the system. In addition, morphine did not alter the response to a synthetic mRNA, polyuridylic acid. However, both the polyribosomes and pH 5 factors isolated from chronically morphinized mouse brain were more effective in promoting amino acid incorporation into protein relative to the corresponding fractions from placebo treated mice. Acrylamide gel electrophoresis of the proteins in the incubation mixture showed the increased amino acid incorporation was the result of a general quantitative increase in the specific activity of all of the proteins synthesized by the cell free system.     

Growth of Thermus aquaticus and its TaqI endonuclease production

The culture behaviour of Thermus aquaticus was characterized. The response of the bacterium to various carbon (tryptone, glucose, glycerol) and nitrogen sources (yeast extract, NaNO₃, (NH₄)₂SO₄, leucine, thymine, thiamine, glutamic acid) was studied. Amino acids did not support growth, but CASTENHOLZ salt medium supplemented with yeast extract and glucose or tryptone resulted in good growth and production. A suitable medium composition giving the highest biomass concentration and enzyme yield was developed. The simple medium containing TYE-NaCl resulted in the highest biomass concentration, whereas CASTENHOLZ mineral medium supplemented with tryptone and yeast extract gave the highest specific activity and enzyme yield. The effect of inoculum age and size on growth was also investigated in order to improve the yield and process consistency. The use of shake flasks inoculated with precultures at their early or late stationary phase resulted in the same biomass concentration (0.56 ± 0.015 g/l) and similar maximum specific growth rates (0.258 ± 0.003 h^?1). Inoculum sizes between 1 and 2.5 per cent were optimal for cell growth. As the other papers on thermophilic microorganisms, including the T. aquaticus YT-1 strain, gave qualitative information on growth, the results presented here cannot be compared with others on a quantitative basis. TaqI endonuclease was purified using a 5 step protocol including cell disruption, adsorption, precipitation, column chromatography and final dialysis. The enriched fraction had a specific activity of 33,600 U TaqI endonuclease per mg protein.     

3-Hydroxybenzoate:coenzyme A ligase and 4-coumarate: coenzyme A ligase from cultured cells of Centaurium erythraea

3-Hydroxybenzoate:coenzyme A ligase, an enzyme involved in xanthone biosynthesis, was detected in cell-free extracts from cultured cells of Centaurium erythraea Rafn. The enzyme was separated from 4-coumarate:coenzyme A ligase by fractionated ammonium sulphate precipitation and hydrophobic interaction chromatography. The CoA ligases exhibited different substrate specificities. 3-Hydroxybenzoate:coenzyme A ligase activated 3-hydroxybenzoic acid most efficiently and lacked affinity for cinnamic acids. In contrast, 4-coumarate:CoA ligase mainly catalyzed the activation of 4-coumaric acid but did not act on benzoic acids. The two enzymes were similar with respect to their relative molecular weight, their pH and temperature optima, their specific activity and the changes in their activity during cell culture growth. Received: 23 September 1996 / Accepted: 28 November 1996     

Characteristics of an Intracellular Proteinase System of a Trichosporon Species Isolated from Trappist-Type Cheese

A greater yield of the active principle was obtained when the cells were allowed to autolyze than when mechanical methods of cell disruption were used. A marked increase in activity was obtained when the crude cell extracts were held for 18 hr at 25 C after acidification to pH 5.0. The activated enzyme was partially purified by precipitation with 95% ethanol. This partially purified extract was most active at pH 5.8, and showed activity over a temperature range of 10 to 42 C. Its rate of activity was markedly decreased by sulfhydryl-binding agents. It exhibited its greatest degree of thermostability at pH 5.0.     

High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters.     

Kinetic study on pH dependence of growth and death of Streptococcus faecalis

A chemostat culture was used for lactic acid fermentation with Streptococcus faecalis at various pH values (8.0, 7.0, 6.0, 5.5, 5.0) and glucose concentrations (10, 20, 30 g/l). At every pH value, the reciprocals of the specific consumption rate of glucose and the specific production rate of lactic acid were linearly correlated to the reciprocal of the specific growth rate. The product, lactic acid, caused non-competitive inhibition of the specific growth rate at every pH value. Moreover, it was found that the cell death rate was dependent on pH and lactic acid. The death rate was smallest at pH 7.0 and increased with increasing lactic acid concentration. The kinetic equations of growth and death are proposed in a broader pH range. Correspondence to: H. Ohara     

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes

Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.     

Simplified Large-Scale Refolding,Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2^nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins.     

Hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC) based facile purification of recombinant streptokinase from E. coli inclusion bodies

The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD(600)=40) culture of recombinant E. coli, and an SK protein concentration of 1080 mg l(-1) was achieved. The wet cell pellet after centrifugation was re-suspended in 8M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1M NaCl during loading, 82.7% rSK activity could be recovered in the 10mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18 x 10(5) in cell lysate to 0.53 x 10(5)IU mg(-1) resulted after this step. rSK was further purified to near-homogeneity (specific activity=0.96 x 10(5)IU mg(-1)) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.     

Microtubule arrays in fucoid zygotes are sensitive to cytoplasmic pH

Regulation of microtubule (MT) arrays and embryo‐genesis by cytoplasmic pH (pH_c) was investigated in zygotes of the brown alga Pelvetia compressa (J. Agardh) De Toni. pH_c was clamped to (set to) acidic values using a weak acid, propionic acid (PA), and to alkaline values using a weak base, methylamine (MA). Acidification of pH_c from the normal value of 7.4–7.5 to about 7.0 caused disruption of microtubule arrays. The nucleating activity was delocalized from the centrosomes and dispersed over the nuclear envelope, the number of MTs decreased, and MTs failed to extend into the cell cortex. Alkalinization to about pH 8.0 also caused dispersal of nucleating activity, but distinct centrosomes remained. MTs coursed in various directions following MA treatment, giving the array a disorganized appearance. Two MT‐dependent processes, rhizoid morphogenesis and cell division, were found to be perturbed by small changes in pH_c.     

Stress proteins in the cytoplasmic membrane fraction of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Stress proteomes of the cytoplasmic membrane fraction of Bacillus subtilis trp _C2-exposed to acid pH and ethanol were characterized. Although these stress factors impair the cell function in a specific manner, they share the ability to denature proteins. Therefore, specific and general stress proteins in the membranes were investigated. Both ethanol (3 %) and pH 5.0 increase the doubling time from 17 to 25 min. Isolated cytoplasmic membranes were subjected to an optimized 2D PAGE analysis which permitted the separation and analysis of ≈450 distinct protein spots. Two alternative methods of protein detection were compared, i.e. silver staining and ³⁵S-l-methionine pulse labeling; the stress induced proteins were identified by MALDI-TOF MS. After ethanol stress, five proteins were increased, viz. YdaP, Ctc, YfhM, YjcH and YwaC. Acid stress proteins were AcoB, YkwC, SodA, YjcH and YwaC. Proteins YjcH and YwaC were increased after ethanol as well as acid pH treatment.