Enhanced pepsin digestion: a novel process for purifying antibody F(ab′)2 fragments in high yield from serum

Jones, R. L.; Landon, J.

doi:10.1016/s0022-1759(02)00031-5

Cited by 65 publications

(47 citation statements)

References 45 publications

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“…Aggressive treatments like pepsin digestion and low pH lead to immunoglobulin denaturation that, in turn, diminishes activity and augments aggregate levels (67)(68)(69). Given that the presence of Fc fragments has been questioned as an important cause of adverse reactions, pepsin digestion of immunoglobulins should be reconsidered as a necessary process step, due to the important increase of aggregate levels achieved.…”

Section: Csa By Protein Aggregatesmentioning

confidence: 99%

“…Given that the presence of Fc fragments has been questioned as an important cause of adverse reactions, pepsin digestion of immunoglobulins should be reconsidered as a necessary process step, due to the important increase of aggregate levels achieved. Despite this, if immunoglobulin digestion is desired, another step to eliminate aggregates should be included (68,69).…”

Section: Csa By Protein Aggregatesmentioning

confidence: 99%

See 1 more Smart Citation

Snake antivenoms: adverse reactions and production technology

Morais¹,

Massaldi²

2009

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

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Antivenoms have been widely used for more than a century for treating snakebites and other accidents with poisonous animals. Despite their efficacy, the use of heterologous antivenoms involves the possibility of adverse reactions due to activation of the immune system. In this paper, alternatives for antivenom production already in use were evaluated in light of their ability to minimize the occurrence of adverse reactions. These effects were classified according to their molecular mechanism as: anaphylactic reactions mediated by IgE, anaphylactoid reactions caused by complement system activation, and pyrogenic reactions produced mainly by the presence of endotoxins in the final product. In the future, antivenoms may be replaced by humanized antibodies, specific neutralizing compounds or vaccination. Meanwhile, improvements in antivenom quality will be focused on the obtainment of a more purified and specific product in compliance with good manufacturing practices and at an affordable cost.

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Section: Csa By Protein Aggregatesmentioning

confidence: 99%

Section: Csa By Protein Aggregatesmentioning

confidence: 99%

Snake antivenoms: adverse reactions and production technology

Morais¹,

Massaldi²

2009

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

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“…4,5 However, since having an even or odd number of cysteines in the hinge region affects the methods used downstream and the resulting final yields, we wanted to characterize in detail the cleavage site(s). We digested a series of mIgG1s with both ficin and pepsin and then analyzed the cleaved products by mass spectrometry and SDS-PAGE.…”

Section: Resultsmentioning

confidence: 99%

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

Patke¹,

Li²,

Wang³

et al. 2017

mAbs

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Bispecific antibodies are a growing class of therapeutic molecules. Many of the current bispecific formats require DNA engineering to convert the parental monoclonal antibodies into the final bispecific molecules. We describe here a method to generate bispecific molecules from hybridoma IgGs in 3-4 d using chemical conjugation of antigen-binding fragments (Fabs) (bisFabs). Proteolytic digestion conditions for each IgG isotype were analyzed to optimize the yield and quality of the final conjugates. The resulting bisFabs showed no significant amounts of homodimers or aggregates. The predictive value of murine bisFabs was tested by comparing the T-cell redirected cytotoxic activity of a panel of antibodies in either the bisFab or full-length IgG formats. A variety of antigens with different structures and expression levels was used to extend the comparison to a wide range of binding geometries and antigen densities. The activity observed for different murine bisFabs correlated with those observed for the fulllength IgG format across multiple different antigen targets, supporting the use of bisFabs as a screening tool. Our method may also be used for the screening of bispecific antibodies with other mechanisms of action, allowing for a more rapid selection of lead therapeutic candidates.

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“…When IgM is digested by pepsin, the molecular weight of the resulting F(ab=) 2 fragments is approximately 100 kDa, smaller than that of IgG. 21 Because monovalent Fab= fragments from the sera of PV patients are capable of inducing blister formation, one can conclude that the Fc portion of IgG does not directly contribute to the pathogenesis of blister formation in pemphigus.…”

Section: Discussionmentioning

confidence: 99%

Pathogenic Relevance of IgG and IgM Antibodies against Desmoglein 3 in Blister Formation in Pemphigus Vulgaris

Tsunoda

Ota

Saito

et al. 2011

The American Journal of Pathology

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Pemphigus vulgaris is an autoimmune disease caused by IgG antibodies against desmoglein 3 (Dsg3). Previously, we isolated a pathogenic mAb against Dsg3, AK23 IgG, which induces a pemphigus vulgaris-like phenotype characterized by blister formation. In the present study, we generated a transgenic mouse expressing AK23 IgM to examine B-cell tolerance and the pathogenic role of IgM. Autoreactive transgenic B cells were found in the spleen and lymph nodes, whereas antiDsg3 AK23 IgM was detected in the cardiovascular circulation. The transgenic mice did not develop an obvious pemphigus vulgaris phenotype, however, even though an excess of AK23 IgM was passively transferred to neonatal mice. Similarly, when hybridoma cells producing AK23 IgM were inoculated into adult mice, no blistering was observed. Immunoelectron microscopy revealed IgM binding at the edges of desmosomes or interdesmosomal cell membranes, but not in the desmosome core, where AK23 IgG binding has been frequently detected. Furthermore, in an in vitro dissociation assay using cultured keratinocytes, AK23 IgG and AK23 IgM F(ab=) 2 fragments, but not AK23 IgM, induced fragmentation of epidermal sheets. Together, these observations indicate that antibodies must gain access to Dsg3 integrated within desmosomes to induce the loss of keratinocyte cell-cell adhesion. These findings provide an important framework for improved understanding of B-cell tolerance and the pathophysiology of blister formation in pemphigus. Pemphigus vulgaris (PV) is a life-threatening, organ-specific autoimmune blistering disease of the skin and mucous membranes. It is characterized clinically by painful oral erosions and flaccid skin blisters, histologically by suprabasal acantholysis (ie, loss of cell-cell adhesion between suprabasal keratinocytes), and immunopathologically by IgG autoantibodies against desmoglein 3 (Dsg3), a cadherin-type cell-cell adhesion molecule found in desmosomes.1,2 Compelling evidence indicates that IgG autoantibodies against Dsg3 are pathogenic and play a primary role in inducing blister formation in pemphigus. IgGs affinity-purified from the sera of PV patients using the extracellular domain of Dsg3 cause suprabasal acantholysis when injected into neonatal mice.3 When anti-Dsg3 IgG is immunoadsorbed from the sera of PV patients using the same Dsg3 domain, those sera lose their ability to cause blister formation in neonatal mice. 4 Furthermore, monoclonal antibodies (mAbs) against Dsg3 from a model mouse and from PV patients induce the formation in mice of blisters with typical PV histology. 5,6 The pathogenic roles of autoantibodies against nondesmoglein molecules remain to be clarified. 7,8 We previously developed a PV model mouse by the adoptive transfer of lymphocytes from Dsg3 Ϫ/Ϫ mice immunized with rDsg3 to Rag2 Ϫ/Ϫ mice that express Dsg3. 9Recipient mice showed stable anti-Dsg3 IgG production and developed a PV phenotype characterized by mucosal erosions and acantholytic blisters, similar to those seen in PV patients. We subsequently isolated AK...

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Enhanced pepsin digestion: a novel process for purifying antibody F(ab′)2 fragments in high yield from serum

Cited by 65 publications

References 45 publications

Snake antivenoms: adverse reactions and production technology

Snake antivenoms: adverse reactions and production technology

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

Pathogenic Relevance of IgG and IgM Antibodies against Desmoglein 3 in Blister Formation in Pemphigus Vulgaris

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