Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration

With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use.     

Selective precipitation-assisted recovery of immunoglobulins from bovine serum using controlled-fouling crossflow membrane microfiltration

Efficient and economic recovery of immunoglobulins (Igs) from complex biological fluids such as serum, cell culture supernatant or fermentation cell lysate or supernatant, represents a substantial challenge in biotechnology. Methods such as protein A affinity chromatography and anion exchange chromatography are limited by cost and selectivity, respectively, while membrane chromatography is limited by low adsorptive area, flow distribution problems and scale-up difficulties. By combining the traditional salt-assisted precipitation process for selective removal of Igs from serum followed by constant-permeate flux membrane microfiltration for low fouling, we demonstrate an exciting new, efficient and economic hybrid method. The high selectivity of an ammonium sulfate-induced precipitation step was used to precipitate the Igs leaving the major undesirable impurity, the bovine serum albumin (BSA), in solution. Crossflow membrane microfiltration in diafiltration mode was then employed to retain the precipitate, while using axial flow rates to optimize removal of residual soluble BSA to the permeate. The selectivity between immunoglobulin G (IgG) and BSA obtained from the precipitation step was approximately 36, with 97% removal of the BSA with diafiltration in 5 diavolumes with resulting purity of the IgG of approximately 93% after the membrane microfiltration step. Complete resolubilization of the IgG was obtained without any aggregation at the concentrations of ammonium sulfate employed in this work. Further, membrane pore size and axial Reynolds number (recirculation rate) were shown to be important for minimizing fouling and loss of protein precipitate.     

Purification of hemoglobin by tangential flow filtration with diafiltration

A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189–199, 2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC‐grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch‐mode diafiltration step to the previously mentioned TFF Hb purification process. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS‐PAGE, and LC‐MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three stage TFF process is sufficient to produce HPLC‐grade bHb. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Extraction of rIL-2 inclusion bodies from Escherichia coli using cross-flow filtration

A cross-flow membrane filtration process was developed for the recovery of rIL-2 inclusion bodies from homogenized Escherichia coli. The membrane extraction process was comprised of a two-step diafiltration followed by an extraction with 7 M GuHCl and a 40-fold dilution of the solubilized inclusion bodies into 0.01 M Tris-HCl, 0.035 M NaCl, pH 7.9. The first diafiltration was with a 0.03 M Tris-HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8, followed by a diafiltration with 1.75 M GuHCl. All of the insoluble rIL-2 was retained behind the membrane, whereas a GuHCl wash solubilized approximately 15% of the rIL-2. The membrane process increased the yield of rIL-2 in the diluted extract by threefold as compared to a similar centrifuge process with a significant increase in purity as determined by reverse-phase high-performance liquid chromatography (HPLC). (c) 1994 John Wiley & Sons, Inc.     

Optimization‐based framework for resin selection strategies in biopharmaceutical purification process development

This work addresses rapid resin selection for integrated chromatographic separations when conducted as part of a high‐throughput screening exercise during the early stages of purification process development. An optimization‐based decision support framework is proposed to process the data generated from microscale experiments to identify the best resins to maximize key performance metrics for a biopharmaceutical manufacturing process, such as yield and purity. A multiobjective mixed integer nonlinear programming model is developed and solved using the ε‐constraint method. Dinkelbach's algorithm is used to solve the resulting mixed integer linear fractional programming model. The proposed framework is successfully applied to an industrial case study of a process to purify recombinant Fc Fusion protein from low molecular weight and high molecular weight product related impurities, involving two chromatographic steps with eight and three candidate resins for each step, respectively. The computational results show the advantage of the proposed framework in terms of computational efficiency and flexibility. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1116–1126, 2017     

A large-scale purification of paclitaxel from cell cultures of Taxus chinensis

A large-scale purification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. The complete method for mass production was a simple and efficient procedure, for the isolation and purification of paclitaxel from the biomass of Taxus chinensis, consisting of solvent extraction, synthetic adsorbent treatment, and two steps of precipitation, followed by two steps of high performance liquid chromatography (HPLC). The organic solvent extraction of biomass obtained crude extract containing paclitaxel. The use of synthetic adsorbent treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimise solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. The paclitaxel of over 99.5% purity can be simply obtained with high yield from crude paclitaxel by HPLC using reverse-phase separation on C18 as the first step and normal-phase separation on silica as the second step.     

gamma-Glutamyl transpeptidase: relation to immunoglobulin A and free secretory component.

The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S-(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p-nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10⁶. The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.     

Whey protein fractionation: isoelectric precipitation of alpha-lactalbumin under gentle heat treatment

The selective precipitation of alpha-lactalbumin (alpha-LA) at a pH around its isoelectric point (4.2) under heat treatment is the basis for a fractionation process of whey proteins. In these conditions, beta-lactoglobulin remains soluble, whereas bovine serum albumin and immunoglobulins co-precipitate. Knowledge of the mechanism governing the alpha-LA precipitation influences the choice of operating conditions and enables optimization of the fractionation process. alpha-LA is a calcium metallo-protein and its isoelectric precipitation is governed by the protein-calcium complexation equilibrium. Citrate, a sequestrant of calcium, decreases the free calcium concentration and displaces the precipitation phenomenon to a lower temperature range. A study of the effect of citrate on the precipitation phenomena of whey proteins is presented. Whatever the citrate content, precipitation curves for bovine serum albumin (BSA) and alpha-LA intersect at a temperature around 45 degrees C. For a temperature of heat treatment lower than 40 degrees C, a selective enrichment in alpha-LA of the precipitated phase is observed. As addition of citrate leads to high alpha-LA precipitated fractions at a temperature around 35 degrees C, the precipitation step may be performed at this temperature. It results in a reduced heat denaturation of whey proteins and in a higher alpha-LA purity in the precipitated fraction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 391-397, 1997.     

抗甲肝病毒基因工程单抗分离纯化与鉴定

为克服血源免疫球蛋白制品的不足,开发了抗甲肝病毒基因工程单克隆抗体anti-HAV IgG。用无血清培养基培养rCHO工程细胞株,上清液经过rProtein A SFF亲和层析→脱盐→离子交换层析→超滤换液纯化后,所得anti-HAV IgG纯度达99%以上,比活性约100IU/mg,anti-HAV IgG活性回收率40%。所纯化的anti-HAV IgG分子量150kD,等电点8.4～9.3。免疫印迹实验证实anti-HAV IgG为人源全抗体分子。亲和层析介质rProtein A SFF确实存在亲和配基脱落问题,但通过后续纯化步骤可有效除去。在亲和层析过程中加入高盐清洗步骤,可有效降低宿主DNA残留量水平。对样品中自由巯基含量进行了测定,认为非还原电泳图谱中低分子量条带是由于抗体分子内存在自由巯基引起。用该工艺制备的anti-HAV IgG各项纯度检测指标均达到我国对基因工程产品的质量要求。     

Process integration for recovery of recombinant collagen type I α1 from corn seed

Because of safety concerns and product consistency issues with the use of animal‐derived collagen, several recombinant protein expression hosts have been considered for recombinant collagen corn seed. Full length, triple‐helical, recombinant collagen (rCIα1) is expressed as a fusion with a foldon domain, which must later be removed. Here we have examined integration of purification and foldon removal by comparing advantages of removal before or after purification, using salt precipitation as the main purification step. Because expression levels in available maize lines are low, Pichia‐produced recombinant collagens, both with and without foldon, were added to corn seed germ at the extraction step. Salt precipitation of an acidic corn seed extract yielded 100% of the collagen without foldon at >70% purity without the pepsin pretreatment. With pepsin pretreatment, yield was 94.0% with purity of 76.5%. Analysis of the protein molecular weight distribution of the pre‐ and post‐treatment extracts showed that the corn proteins are largely resistant to pepsin proteolysis, explaining why little benefit was obtained by pepsin treatment. In the absence of pepsin treatment, the recovery of rCIα1 with foldon was still above 90% but the purity was only 44%. This still represented at about 13‐fold purification with a 2.7‐fold volume reduction which would reduce the pepsin requirement for post‐recovery foldon cleavage. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:98–107, 2016     

Use of ultrafiltration/diafiltration for the processing of antisense oligonucleotides

Ultrafiltration/diafiltration (UF/DF) has been the hallmark for concentrating and buffer exchange of protein and peptide-based therapeutics for years. Here we examine the capabilities and limitations of UF/DF membranes to process oligonucleotides using antisense oligonucleotides (ASOs) as a model. Using a 3 kDa UF/DF membrane, oligonucleotides as small as 6 kDa are shown to have low sieving coefficients (<0.008) and thus can be concentrated to high concentrations (≤200 mg/mL) with high yield (≥95%) and low viscosity (<15 centipoise), provided the oligonucleotide is designed not to undergo self-hybridization. In general, the oligonucleotide should be at least twice the reported membrane molecular weight cutoff for robust retention. Regarding diafiltration, results show that a small amount of salt is necessary to maintain adequate flux at concentrations exceeding about 40 mg/mL. Removal of salts along with residual solvents and small molecule process-related impurities can be robust provided they are not positively charged as the interaction with the oligonucleotide can prevent passage through the membrane, even for common divalent cations such as calcium or magnesium. Overall, UF/DF is a valuable tool to utilize in oligonucleotide processing, especially as a final drug substance formulation step that enables a liquid active pharmaceutical ingredient.     

Downstream processing of high chain length polysialic acid using membrane adsorbers and clay minerals for application in tissue engineering

Polysialic acid (polySia) is a carbohydrate polymer of varying chain length. It is a promising scaffold material for tissue engineering. In this work, high chain length polySia was produced by an Escherichia coli K1 strain in a 10‐L bioreactor in batch and fed‐batch mode, respectively. A new downstream process for polySia is presented, based on membrane adsorber technology and use of inorganic anion exchanger. These methods enable the replacement of precipitation steps, such as acetone, cetavlon, and ethanol precipitation of the already established purification process. The purification process was simplified, while process efficiency and product qualities were improved. The overall yield of polySia from a 10‐L batch cultivation process was 61% and for 10‐L fed‐batch cultivation process the yield was 40% with an overall purity of 98%. The endotoxin content was determined to be negligible (14 EU mg^?1). The main advantage of this new downstream process is that polySia with high chain length of more than 130 degree of polymerization can be obtained. In fed‐batch cultivation, chain lengths up to 160 degree of polymerization were obtained.     

Assessing changes in Cd phytoavailability to tomato in amended calcareous soils

An efficient production method of heme-iron-enriched peptide was developed based on enzymatic hydrolysis. Hemoglobin hydrolysis, carried out stepwise with commercially available exopeptidase and endopeptidase, resulted in an increased degree of hydrolysis (DH). Exopeptidase-catalyzed protein hydrolysis formed low molecular weight peptides and amino acids. Different process parameters including dialysis and ultra- and diafiltration were evaluated. Heme/peptide ratio increased as molecular weight cut-off (MWCO) of the dialysis membrane increased. When the hydrolysate was dialyzed against sodium phosphate buffer, a higher heme/ peptide ratio was obtained. The heme/peptide ratio of the hydrolysate reached up to 25.4% when the dialysis was carried out with a membrane of 12-14 kDa MWCO. Also, the ratio was improved by the use of ultrafiltration and diafiltration on the pilot-scale.     

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     

Recovery of polyhydroxybutyrate (PHB) from Cupriavidus necator biomass by solvent extraction with 1,2‐propylene carbonate

An integrated procedure for the recovery of polyhydroxybutyrate (PHB) produced by Cupriavidus necator based on the extraction with 1,2‐propylene carbonate was evaluated. The effect of temperature (100–145°C) and contact time (15–45 min), precipitation period, and biomass pretreatments (pH shock and/or thermal treatments) on PHB extraction efficiency and polymer properties was evaluated. The highest yield (95%) and purity (84%) were obtained with the combination of a temperature of 130°C and a contact time of 30 min, with a precipitation period of 48 h. Under these conditions, PHB had a molecular weight of 7.4×10⁵, which was the highest value obtained. Lower values (2.2×10⁵) were obtained for higher temperatures (145°C), while lower temperatures resulted in incomplete extraction yields (45–54%). No further yield improvement was achieved with the pH/heat pretreatments, but the polymer's molecular weight was increased to 1.3×10⁶. The PHB physical properties were not significantly affected by any of the tested procedures, as shown by the narrow ranges obtained for the glass transition temperature (4.8–5.0°C), melting temperature (170.1–180.1°C), melting enthalpy (77.8–88.5 J/g) and crystallinity (55–62%). 1,2‐Propylene carbonate was shown to be an efficient solvent for the extraction of PHB from biomass. The precipitation procedure was found to highly influence the polymer recovery and its molecular weight. Although polymer molecular weight and purity were improved by applying pH/heat pretreatment to the biomass, the procedure involves the use of large amounts of chemicals, which increases the recovery costs and makes the process environmentally unfriendly.     

大豆乳清中蛋白质和异黄酮的超滤分离技术

研究了用超滤技术分离大豆乳清中的蛋白质和异黄酮的工艺条件. 结果表明,大豆乳清在超滤之前要进行预处理以减轻膜污染;通过2因素3水平正交试验,确定了最佳的预处理工艺：按大豆乳清中固形物含量的5%,向其中加入CaCl₂,并在85 ℃下加热15 min,在此条件下,蛋白质的沉淀率为49.8%,异黄酮的保留率为90.4%;通过单因素试验,确定了比较合适的超滤条件：选择切割分子量（MWCO）为10000的聚醚砜膜,超滤压力选择51～68 kPa,超滤温度选择30 ℃～40 ℃.在此条件下,大豆乳清中蛋白质的截留率为83.9%,而异黄酮的截留率为7.6%.     

High‐throughput process development of purification alternatives for the protein avidin

With an increased number of applications in the field of the avidin‐biotin technology, the resulting demand for highly‐purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high‐throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion‐exchange chromatography. In a high‐throughput format, process parameters for aqueous two‐phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed‐mode column chromatography experiments were performed. The HTPD strategy was complemented by a high‐throughput tandem high‐performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two‐phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two‐phase extraction and precipitation results were largely confirmed in larger scale with scale‐up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed‐mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:957–973, 2015     

Separation of alpha-lactalbumin and beta-lactoglobulin using membrane ultrafiltration

There is considerable commercial interest in the preparation of individual whey proteins as high-value food additives, nutraceuticals, and therapeutics. This study examined the use of membrane filtration for the separation of alpha-lactalbumin and beta-lactoglobulin. Stirred cell filtration experiments were performed using both cellulosic and polyethersulfone membranes to determine the optimal pH, ionic strength, and filtration conditions. Selectivities of greater than 55 could be achieved at pH 5.5 and 50 mM ionic strength using a 30-kD cellulose membrane. A diafiltration process was then designed for the protein separation. A 16-diavolume filtration yielded beta-lactoglobulin as the retentate product with a purification factor of 100 and recovery of 90%. The alpha-lactalbumin was recovered in the filtrate with a purification factor of more than 10 and nearly 99% yield. Model calculations were in good agreement with the experimental data.     

Highly selective synthesis of 1,3-oleoyl-2-palmitoylglycerol by lipase catalysis.

1,3-Oleoyl-2-palmitoylglycerol (OPO), an important structured triglyceride in infant nutrition, was synthesized by a two-step process in high yields and purity using sn1,3-regiospecific lipases. In the first step, tripalmitin (TP) was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn1,3-regiospecific lipases yielding the corresponding 2-monopalmitin (2-MP). The 2-MP was isolated in up to 85% yield and >95% purity by crystallization and esterified in the second step with oleic acid using the same lipases to form the structured triglyceride OPO in up to 78% yield containing 96% palmitic acid in the sn2-position. Water activity, solvent, as well as carrier for lipase immobilization strongly influenced the yield and purity of products in both steps. The best results were achieved with lipases from Rhizomucor miehei and Rhizopus delemar immobilized on EP 100 and equilibrated to a water activity of 0.43. Special emphasis was given to develop this process in solvents that are allowed to be used in foodstuffs and to perform the second step in a solvent-free system.     

Purification of Monomeric Prolactin Charge Isoform from Buffalo Pituitaries

Abstract

A standard protocol for isolation of buffalo prolactin (buPRL) was modified at the alcohol precipitation step. This modification could separate lower molecular weight prolactin from the higher molecular weight prolactin (PRL). Reloading the prolactin onto a Sephacryl S‐200 gel purified the buPRL monomer. The purity of buPRL monomer was confirmed by 15% SDS PAGE. The buPRL monomer was >90% pure. It was characterized by specific anti‐buPRL serum in ELISA and Western blot. A native PAGE of the PRL showed three charge isoforms. A protocol was standardized to separate prolactin monomeric least acidic isoforms using an anion exchanger.