Single-chain antibody fragments: Purification methodologies

At present, single-chain variable fragments (scFv) of antibodies are considered one of the most important tools in human therapies. Wide applications of antibodies are being exploited in different medical, pharmaceutical and research areas. These molecules maintain the same binding functionality that full length antibodies but possess several advantageous features as quickness to penetrate the tissues, easy manipulation, fast elimination of their immunocomplex and the possibility of being produced in simple expression systems like bacteria and yeast. The increasing demand in antibody based methodologies is driving advances in the production and purification of genetically engineered antibodies and antibody fragments. While advances in expression systems allow the production of high titers of antibodies, there exist some limits imposed by the downstream methodologies which are not efficient enough to ensure their industrialization.The main aim of this review is to highlight the principal characteristics of single-chain variable fragments of antibodies addressing advances and perspectives on scFv purification.     

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

Relationship between the protein surface hydrophobicity and its partitioning behaviour in aqueous two-phase systems of polyethyleneglycol-dextran

In order to develop possible correlations to predict partioning behaviour of proteins, five mammalian albumins (goat, bovine, equine, human and pig ones) with similar physico-chemical properties (molecular mass and isoelectrical point) were chosen. Evaluation of the relationship between hydrophobicity and partitioning coefficient (Kr) in polyethylenglycol-dextran (PEG-DxT500) systems formed by polyethyleneglycols of different molecular mass (3350, 6000 and 10,000) was investigated by estimating relative surface hydrophobicity (So) with a fluorescent probe, 1 anilino-8-naphthalene sulfonate. No relationship between Kr and So was found for systems formed by PEG3350, while aqueous two-phase systems with PEG6000 and PEG10,000 gave better correlations. The results obtained may be explained on the basis of an increase in the interaction between the latter PEGs and the protein due to their higher hydrophobic character which increases as the PEG molecular mass does so. In this way, systems with PEGs of higher molecular mass give the highest resolution to exploit hydrophobicity in partitioning.     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.     

Role of polymer–protein interaction on partitioning pattern of bovine pancreatic trypsinogen and alpha-chymotrypsinogen in polyethyleneglycol/sodium tartrate aqueous two-phase systems

The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5–1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG–protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.     

Protein-flexible chain polymer interactions to explain protein partition in aqueous two-phase systems and the protein-polyelectrolyte complex formation

Complexes formation between two model proteins (catalase and chymotrypsin) and polyelectrolytes (polyvinyl sulphonate and polyacrilic acid) and a non-charged flexible chain polymer (PCF) as polyethylene propylene oxide (molecular mass 8400) was studied by a spectroscopy technique combination: UV absorption, fluorescence emission and circular dichroism. All the polymers increase the protein surface hydrophobicity (S(0)) parameter value as a proof of the modification of the protein surface exposed to the solvent. Chymotrypsin showed an increase in its biological activity in polymer presence, which suggests a change in the superficial microenvironment. The decrease in the biological activity of catalase might be due to a competition between the polymer and the substrate. This result agrees with the polymer effect on the catalase superficial hydrophobic area. It was found that, when flexible chain polymers increase protein stability and the enzymatic activity they could be used to isolate this enzyme without inducing loss of protein enzymatic activity. Our findings suggest that the interactions are dependent on the protein physico-chemical parameters such as: isoelectric pH, hydrophobic surface area, etc.     

The ITGAV rs3738919 variant and susceptibility to rheumatoid arthritis in four Caucasian sample sets

Introduction

Angiogenesis is an important process in the development of destructive synovial pannus in rheumatoid arthritis (RA). The ITGAV +gene encodes a cell cycle-associated antigen, integrin ανβ 3, which plays a role in RA angiogenesis. Previously, two independent studies identified an association between the major allele of the ITGAV single-nucleotide polymorphism (SNP) rs3738919 and RA. We therefore tested this association in an independent study using New Zealand (NZ) and Oxford (UK) RA case control samples.

Methods

We compared genotype frequencies in 740 NZ Caucasian RA patients and 553 controls genotyped for rs3738919, using a polymerase chain reaction-restriction fragment length polymorphism assay. A TaqMan genotyping SNP assay was used to type 713 Caucasian RA patients and 515 control samples from Oxford for the rs3738919 variant. Association of rs3738919 with RA was tested in these two sample sets using the chi-square goodness-of-fit test. The Mantel-Haenszel test was used to perform a meta-analysis, combining the genetic results from four independent Caucasian case control cohorts, consisting of 3,527 cases and 4,126 controls. Haplotype analysis was also performed using SNPs rs3911238, rs10174098 and rs3738919 in the Wellcome Trust Case Control Consortium, NZ and Oxford case control samples.

Results

We found no evidence for association between ITGAV and RA in either the NZ or Oxford sample set (odds ratio [OR] = 0.88, P_allelic = 0.11 and OR = 1.18, P_allelic = 0.07, respectively). Inclusion of these data in a meta-analysis (random effects) of four independent cohorts (3,527 cases and 4,126 controls) weakens support for the hypothesis that rs3738919 plays a role in the development of RA (OR_combined = 0.92, 95% confidence interval 0.80 to 1.07; P = 0.29). No consistent haplotype associations were evident.

Conclusions

Association of ITGAV SNP rs7378919 with RA was not replicated in NZ or Oxford case control sample sets. Meta-analysis of these and previously published data lends limited support for a role for the ITGAV in RA in Caucasians of European ancestry.     

Assessment of the effect of triton X‐114 on the physicochemical properties of an antibody fragment

The effect of Triton X‐114 on the physicochemical properties of a single‐chain antibody fragment (scFv) has been studied. According to the far UV circular dichroism spectroscopy, the secondary structure of the recombinant antibody was not significantly affected by the presence of Triton. From the antibody tertiary structure analysis, it was found that the surfactant could be located around the tryptophan molecules accessible to the solvent, diminishing the polarity of its environment but maintaining most of the protein structure integrity. However, in certain conditions of high temperature and high concentration of denaturant molecules, the presence of TX could compromise the antibody fragment stability. These results represent a previous step in designing scFv purification protocols and should be considered prior to developing scFv liquid–liquid extraction procedures. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:554–561, 2014     

Partitioning features of bovine trypsin and alpha-chymotrypsin in polyethyleneglycol-sodium citrate aqueous two-phase systems

The partitioning of bovine trypsin and alpha-chymotrypsin--proteases of similar physico-chemical properties--in different polyethyleneglycol/sodium citrate aqueous two-phase systems was investigated. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length, temperature and the presence of an inorganic salt on the protein partition coefficient were analysed. Both a decrease in PEG molecular weight and an increase in pH led to a higher partition coefficient for both enzymes. Aqueous two-phase systems formed by PEG of molecular weight 3350 and citrate pH 5.2 showed the best separation capability which was enhanced in presence of sodium chloride 3%. The transfer of both proteins to the top phase was associated with negative enthalpic and entropic changes.     

Porcine pancreatic lipase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

This research study examined porcine pancreatic lipase partition in aqueous two-phase systems formed by polyethylene glycol-potassium phosphate at pH 6.0, 7.0 and 8.0, the effect of polymer molecular mass, and NaCl concentration. The enzyme was preferentially partitioned into the polyethylene glycol rich phase in systems with molecular mass 4000-8000, while with polyethylene glycol of 10,000 molecular mass it was concentrated in the phosphate rich phase. The enthalpic and entropic changes found due to the protein partition were negative for all the polyethylene glycol molecular mass systems assessed. Both thermodynamic functions were shown to be associated by an entropic-enthalpic compensation effect suggesting that the water structure ordered in the ethylene chain of polyethylene glycol plays a role in the protein partition. The addition of NaCl increased the lipase affinity to the top phase and this effect was most significant in the system polyethylene glycol 2000-NaCl 3%. This system yielded an enzyme recovery more than 90% with a purification factor of approximately 3.4.