Immunoglobulin purification by caprylic acid.

At laboratory scale, several methods for the purification of immunoglobulins from plasma or serum are available. However, not all of them are equally applicable when the scale-up to the level of the pharmaceutical industry is intended. In this case, among other factors, it must be taken into account the performance and the cost and quality of the end product. Here we present a method of purification based on the differential precipitation of plasma proteins with caprylic acid in a single step that is simple and cheap and can be easily scaled up. This methodology has been successfully applied to the development and production of pharmaceutical product, such as therapeutic antisera where immunoglobulin fraction is the unique active pharmaceutical ingredient.

Evolution of venom antigenaemia and antivenom concentration in patients bitten by snakes in Uruguay.

In this work we describe the first study carried out in Uruguay of venom antigenaemia and antivenom concentration in patients bitten by snakes. Between 50 and 70 snake bite accidents per year are caused in Uruguay by 2 species: Rhinocerophis alternatus and Bothropoides pubescens. The patients are treated with a specific polyvalent antivenom. Gaining insight on the evolution of venom antigenaemia and antivenom concentration in patients is important to improve treatment protocols. Blood samples of 29 patients were analysed to determine venom and antivenom concentrations at different times. Venom was detected in 18 of 19 samples before antivenom administration, with a mean concentration of 57 ng/mL. Most of the patients received 4 or 8 vials to neutralize the venom effects. Only one patient needed a total of 16 vials. He showed a severe envenomation and needed supplementary amounts of antivenom after the fifth day of the snake bite accident to reach normal clotting parameters. Antivenom concentrations were determined at 12 h, 24 h and 15 days after antivenom administration. It was found a faster antivenom decrease between 12 and 24 h than to 24 h to 15 days. This was explained by a different clearance mechanism in each period. In the first phase, the cause would be the neutralization of venom present in the blood whereas in the second phase it would be due to unbound antivenom elimination.

A model mechanism for protein precipitation by caprylic acid: application to plasma purification.

A model for the mechanism of protein precipitation by caprylic acid (CA) is developed on the basis of quantitative assays of precipitation with bovine serum albumin (BSA) and CA at different concentrations. It was found that the effect of CA is due to direct interaction with the precipitating protein. Maximum precipitation was achieved when the mass ratio of CA-BSA was close to 1, equivalent to about 450 CA molecules per molecule of BSA. This value was confirmed by optimizing the CA purification of immunoglobulins from equine blood plasma. With a sample diluted 1:1, it was found that CA at a final concentration of 3.5% is optimal to obtain immunoglobulins essentially free of albumin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is proposed that CA binds to specific sites of the protein, thereby inducing partial unfolding of the protein, which exposes additional binding sites. More CA molecules incorporate into all sites in the form of mixed micelles. Thus, the interfacial protein surface becomes highly hydrophobic and increases protein-protein attraction, causing association and precipitation of the macromolecular complexes.

Humoral immune responses to venom and antivenom of patients bitten by Bothrops snakes.

Snake envenomation and its treatment cause the entry of two kind of foreign antigens into the human body: snake toxins and antivenom from animal origin. Samples of patients bitten by snakes in Uruguay were assayed to determine levels of human antibodies against venom and antivenom. The ELISA results showed that most of the patients presented an important increase of IgG and IgM antibodies against antivenom at day 15 post accident. Antibodies were reactive against both equine immunoglobulin chains by western blot assay. In the case of the response against the venom, increase in titre at day 15 was of a minor degree as compared with the antivenom by ELISA. Only one of the patients showed an important increase of IgG and IgM levels against Bothropoides pubescens and only of IgG level against Rhinocerophis alternatus. This patient also showed an extensive reactivity against B. pubescens by western blot.

Features of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14.

The effect of several cultivation conditions on the kinetics of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14 was studied. The presence in the supernatant of serotype-specific CPS (capsular polysaccharide) during growth was followed by size-exclusion HPLC and, in parallel, confirmed by using a specific latex reagent. The agitation level did not affect the production behaviour, whereas pH maintenance above 6 strongly enhanced both growth and CPS production throughout the cultivation period in flasks. Production of high-molecular-mass polysaccharide was found to be maximal between 5 and 6 h of cultivation, at the end of the exponential phase. By laser light scattering, 90% of this purified CPS product showed a M(w) (molecular mass) range from 350 to 1500 kDa, with an average M(w) of 921 kDa. Extending the culture to 24 h gave rise to a clear shift of the M(w) distribution of the polysaccharide to values lower than 100 kDa. These findings may have strong implications for the large-scale manufacture of the polysaccharide and the associated conjugate vaccine.

Evolution of endotoxin contamination during production of a therapeutic serum.

A comparative bench-scale study of endotoxin contamination is presented for two common processes of immunoglobulin purification from equine plasma: ammonium sulphate fractionation of F(ab')2 fragments and caprylic acid precipitation of non-IgG proteins. To this end, both processes were carried out under normal sterile conditions, using sanitized material and equipment and optimal water quality in a clean but open environment. Stream samples, taken at different stages from each process, were analyzed for endotoxin content by the Limulus Amebocyte Lysate (LAL) test. It was found that exogenous contamination preferentially came from endotoxins already present in reagents and/or raw materials, whereas contamination from the environment was minimal. Endogenous endotoxin accumulation, concomitant with the concentration of proteins during processing, was found to be an important factor. With classic technology, blood extraction and sterilizing filtration are critical points for both processes. It is concluded that sterility is not a sufficient condition to obtain an endotoxin-free product. Only with proper sanitization of material, and by applying the caprylic acid purification process with a starting plasma below 4-5 EU/mL, would it be possible to achieve a final product within the norm.

Effect of pepsin digestion on the antivenom activity of equine immunoglobulins.

Enzyme digestion of animal-derived sera followed by antibody purification is a classical process used to prepare snake antivenoms worldwide. In this work, we have studied the effect of the harsh conditions prevailing during the digestion step on the activity of the final product, F(ab')(2). To this purpose, the recovery of the activity of anti-Bothrops hyperimmune equine plasma was determined after pepsin digestion under different sets of processing conditions. The balance between pH level and reaction time was found to be critical, reflecting a compromise between complete cleavage of immunoglobulins and strong denaturation of the F(ab')(2) fragments. For pH in the range 2.8-3.2, 30-65% of the initial activity was lost depending mainly on the processing time, as determined by a competition ELISA technique. Pepsin digestion was also carried out with purified immunoglobulins from the same plasma. SDS PAGE run on the digested immunoglobulins allowed us to verify that the lightest isotypes were more resistant to digestion than the heavier ones. In conclusion, for equine F(ab')(2) antivenom production, it seems convenient to carry out digestion at pH values sufficiently low to ensure that total IgG breakdown is achieved in the shortest time compatible with precise operation in the production scale.

Construction and expression of recombinant streptolysin-o and preevaluation of its use in immunoassays.

Commercially available immunoassays for assessment of anti-streptolysin-O antibodies use native streptolysin-O obtained by a complex process. We prepared a biologically active recombinant streptolysin-O with higher yield and a simpler purification process. An enzyme-linked immunosorbent assay developed with this recombinant showed good correlation with a commercial test, suggesting that it could be suitable for immunoassays.

Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation.

Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5+/-3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity.

Purification of capsular polysaccharide from Streptococcus pneumoniae serotype 23F by a procedure suitable for scale-up.

Streptococcus pneumoniae is a pathogenic encapsulated bacterium, which causes pneumonia, bacteraemia and meningitis. Capsular polysaccharide conjugated to a carrier protein has been widely used as a vaccine antigen. Serotype 23F is one of the prevalent worldwide pneumococci. A simple and efficient method for capsular polysaccharide serotype 23F purification that can easily be scaled-up was developed. This method consisted of using culture broth obtained by tangential microfiltration through a 0.22 microm membrane, broth microfiltrate concentration by tangential ultrafiltration in a 30 kDa spiral membrane, fractional ethanol precipitation (28-60%), nuclease and proteinase treatment, and concentration/diafiltration in a 30 kDa cassette membrane. The final polysaccharide recovery was 89%. The final protein and nucleotide contamination was 1.5% (w/w) and 0.3% (w/w) respectively. The final pure polysaccharide meets the requirements of the World Health Organization and residual proteinase was not found in the final product.

Quantitative determination of pneumococcal capsular polysaccharide serotype 14 using a modification of phenol-sulfuric acid method.

The capsular polysaccharide of Streptococcus pneumoniae, serotype 14, is part of every pneumococcal vaccine presently in the market or under development. A strategy for the quantitative determination of this polysaccharide by the phenol-sulfuric acid method is described. The modality of acid addition is shown to be the critical step for obtaining reproducible test results between different technicians. Raising the incubation temperature above 80 degrees C increased the consistency of the method by more than 60% regardless of the acid addition modality, but at the expense of some loss of sensitivity. Incubation at 110 degrees C was found necessary to obtain reproducible results within 3% for this technique, which was used to follow the enrichment of the polysaccharide during the last steps of purification. A model mixture of the component polysaccharide sugars provided an adequate and economic standard to construct the calibration curve for this assay, with absorbance reading either in the reaction tubes or in a microplate. A similar procedure may be applied to the determination of other bacterial polysaccharides as well.