Single-reagent one-step procedures for the purification of ovine IgG, F(ab′)2 and Fab antivenoms by caprylic acid

Al-Abdulla, Ibrahim; Casewell, Nicholas R.; Landon, J.

doi:10.1016/j.jim.2013.11.001

Cited by 14 publications

(18 citation statements)

References 32 publications

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“…Artificial tears, anti-inflammatory drops, and punctal plugs are conventional treatments, but they provide only partial benefits in addition of having side effects. Eye drops made of human serum have emerged as an alternative and probably superior treatment of DES [ 6 , 11 , 38 , 39 ]. SED preparations from autologous sources have so far been used most frequently [ 5 , 6 , 14 , 15 ], and standardized preparation procedures using a closed cascade filtration system were developed [ 40 ].…”

Section: Discussionmentioning

confidence: 99%

“…Serum eye drops (SEDs) made from human blood have recently emerged as an alternative and apparently more-efficient method than conventional treatment to improve the ocular surface health, tear film stability, and subjective comfort in refractory DES [ 5 – 11 ]. The therapeutic benefits of SEDs are probably multifactorial, and can be explained by a composition that shares similarities with that of tears [ 5 ], including antimicrobial components, various platelet-derived growth factors, fibronectin, and vitamins [ 3 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

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Solvent/Detergent Virally Inactivated Serum Eye Drops Restore Healthy Ocular Epithelium in a Rabbit Model of Dry-Eye Syndrome

et al. 2016

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Application of autologous serum eye drops (SEDs) is a recognized means to treat severe dry-eye syndrome (DES). Due to the inconvenience and difficulty of preparing SEDs from some patients, producing SEDs from allogeneic blood donations is gaining popularity. A major safety concern associated with allogeneic blood is virus transmission. We therefore herein evaluated the possibility of applying a solvent/detergent (S/D) treatment to inactivate viruses and studied the impacts of such treatment of SEDs to resolve DES in a rabbit model. Sera prepared from the blood of five rabbits were pooled and divided into two sub-pools. One was untreated (SEDs), while the other was virally-inactivated with 1% Tri-n-butyl phosphate/1% Triton X-45 at 31°C for 1 h (S/D-SEDs). DES was induced in rabbits using 0.1% benzalkonium chloride (BAC). Rabbits were divided into five groups of two rabbits each. One group was untreated (control), three were treated twice daily for 3 weeks using PBS, SEDs, or S/D-SEDs, and the last received an additional 0.1% BAC (as the negative control). The DES condition was determined by measuring aqueous tear secretion (Schirmer’s test), corneal fluorescein staining, a corneal histologic examination, TUNEL stain apoptosis, and corneal inflammatory marker (tumor necrosis factor-α, interleukin (IL)-1β, IL-8, and IL-6) expressions. We first confirmed that SEDs and S/D-SEDs had similar protein profiles and transforming growth factor (TGF)-β contents. Animal experiments showed that tear secretion did not significantly differ between the SED and S/D-SED groups but was significantly higher than in the PBS group. Eye fluorescein staining revealed dramatic improvements in epithelial defects in groups treated with SEDs or S/D-SEDs, and hematoxylin/eosin staining revealed microscopic epithelial layers similar to those of the untreated controls. Inflammatory markers and TUNEL studies showed that healthy epithelium had been restored in groups treated with SEDs or S/D-SEDs. In conclusion, this preclinical study supports the possibility of using S/D virally inactivated SEDs to treat DES and restore a normal epithelium.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Solvent/Detergent Virally Inactivated Serum Eye Drops Restore Healthy Ocular Epithelium in a Rabbit Model of Dry-Eye Syndrome

et al. 2016

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“…One of the most widely used approaches for antivenom production as the initial step employs a salting-out procedure [ 3 - 7 ], which is associated with a low-purity profile of the final product or excessive formation of aggregates [ 8 , 9 ]. Caprylic acid as an alternative precipitating agent [ 10 ] has been introduced into the preparation of a whole series of equine or ovine IgG- or F(ab') 2 -based antivenoms [ 11 - 17 ]. Purification principles based on caprylic acid have been proven more beneficial than salt-mediated methods due to selective precipitation of non-IgG plasma proteins [ 18 , 19 ].…”

Section: Introductionmentioning

confidence: 99%

“…Some antivenom manufacturers perform enzyme-mediated removal of the Fc portion from IgG molecules that can be accomplished upon their purification [ 20 ], on unfractionated plasma [ 16 , 20 , 21 ] or simultaneously with the precipitation of unwanted proteins using caprylic acid, as demonstrated on ovine serum [ 17 ]. Aside from the doubtful role of the Fc fraction in adverse reactions [ 18 , 19 , 22 , 23 ], its removal reduces the quantity of foreign material in the product intended for use in humans, thus increasing specific activity and at least partially contributing to safety improvement.…”

Section: Introductionmentioning

confidence: 99%

Streamlined downstream process for efficient and sustainable (Fab')2 antivenom preparation

Kurtović

Brgles

Balija

et al. 2020

J. Venom. Anim. Toxins incl. Trop. Dis

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Background: Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab') 2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab') 2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio ( w / w ) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab') 2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab') 2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab') 2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.

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“…We selected this treatment for three main reasons. First, caprylic acid is convenient, as it precipitates essentially all non-Ig proteins in a single step, yielding a supernatant containing not only a physiological proportion of all Ig classes and subclasses [ 22 , 32 – 34 ], but also anti-HPA-1a Igs, as revealed by PAK-12 and MAIPA data. Second, caprylic acid is already used to prepare therapeutic Igs, such as horse-plasma derived antivenoms [ 35 , 36 ], and human normal or hyperimmune Igs [ 21 , 37 , 38 ].…”

Section: Discussionmentioning

confidence: 99%

Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia

et al. 2016

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Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements opening perspectives for the prevention of FNAIT.

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Single-reagent one-step procedures for the purification of ovine IgG, F(ab′)2 and Fab antivenoms by caprylic acid

Cited by 14 publications

References 32 publications

Solvent/Detergent Virally Inactivated Serum Eye Drops Restore Healthy Ocular Epithelium in a Rabbit Model of Dry-Eye Syndrome

Solvent/Detergent Virally Inactivated Serum Eye Drops Restore Healthy Ocular Epithelium in a Rabbit Model of Dry-Eye Syndrome

Streamlined downstream process for efficient and sustainable (Fab')2 antivenom preparation

Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia

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