Induction and analysis of aggregates in a liquid IgG1-antibody formulation

Mahler, Hanns‐Christian; Müller, Renate; Frieß, Wolfgang; Delille, Aurelie; Matheus, Susanne

doi:10.1016/j.ejpb.2004.12.004

Cited by 269 publications

(216 citation statements)

References 35 publications

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“…Disparity in the number of anti-HLA IgG Abs recognized by the two 2nd-Abs in sera of normal donors. Table I shows subjected to freeze-thaw and/or centrifugation self-aggregate or aggregate with IgM, complement, and other proteins (9)(10)(11)(12)(13)(14)(15). The polyclonal IgHPolyFab can easily recognize IgH in these aggregates.…”

Section: Disparity In the Serum Anti-hla Igg Profiles And Mfis Betweementioning

confidence: 99%

“…Consistency and specificity of MFI values of serum IgG Abs may be affected by extrinsic factors like IgM (11,12) and aggregation of serum IgG, with or without other serum proteins (13)(14)(15)(16)(17) when neat or low-dilution sera are used (11), and by intrinsic factors, such as the Luminex platform and the vendor-supplied SAB analytical reagents (7,14). Therefore, quality control maximizing the specificity and reliability of MFIs of serum IgG requires serious and step-wise validation of the Ag-coated beads and the detection system.…”

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confidence: 99%

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Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss

Ravindranath

Jucaud

Banuelos

et al. 2017

The Journal of Immunology

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Luminex multiplex immunoassays enable simultaneous monitoring of Abs against multiple Ags in autoimmune, inflammatory, and infectious diseases. The assays are used extensively to monitor anti-HLA Abs in transplant patients for donor organ selection, desensitization, and assessing the risk for graft rejection. To monitor IgG Abs, fluoresceinated IgG constant H chain–binding polyclonal F(ab′)2 (IgHPolyFab) is used as the fluoresceinated secondary Ab (2nd-Ab), whereas IgG subclasses are monitored with Fc-specific monoclonal whole IgG (FcMonoIgG). The fluorescent signal from the 2nd-Ab is measured as mean florescence intensity (MFI). When IgHPolyFab is used, the signal is amplified as a result of the binding of multiple polyclonal Fabs to the C region of primary IgH. The reliability of such amplification for Ab measurements was not validated, nor were MFIs compared with 1:1 binding of FcMonoIgG to primary Abs. Comparing the MFIs of anti-HLA Abs obtained with IgHPolyFab and FcMonoIgG against normal human sera, IVIg, and allograft recipients’ sera, it was observed that the number of HLA-Abs was notably higher with IgHPolyFab than with FcMonoIgG. The MFIs of anti-HLA Abs also remained higher with IgHPolyFab in the normal sera and in IVIg, but the reverse was true when the autologous and allogeneic IgG concentrations were augmented in allograft recipients. Indeed, MFIs of the de novo allo-HLA Abs were markedly higher with FcMonoIgG than with IgHPolyFab. Serum titration established the superiority of FcMonoIgG for monitoring MFIs of de novo allo-HLA Abs in allograft recipients. Avoiding false amplifications of the number and MFIs of anti-HLA IgG with FcMonoIgG may minimize immunosuppressive therapies, maximize the number of donors for patients waiting for allografts, and enable better prediction of graft rejection.

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Section: Disparity In the Serum Anti-hla Igg Profiles And Mfis Betweementioning

confidence: 99%

mentioning

confidence: 99%

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss

Ravindranath

Jucaud

Banuelos

et al. 2017

The Journal of Immunology

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“…15,16 In this study, we used the monoclonal IgG1 antibody Rituximab 17 as a model system to better understand the mechanisms leading to the formation of large aggregates when incubating a multidomain protein under weak thermal stress. Aggregation of antibodies due to various kinds of stress have been addressed in a number of studies, [18][19][20][21][22][23][24] with a few key observations highlighted later. In this context, we distinguish between the formation of oligomers (dimers, trimers, etc.)…”

Section: Introductionmentioning

confidence: 99%

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

et al. 2010

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Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lagtime behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size-exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.

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“…Typically, surfactants have been added to protect against mechanical stresses, namely freeze-thaw and shake. [39][40][41] In addition, surfactants have been successfully used to minimize adsorption to surfaces via competitive binding. 42,43 A review of the current landscape of mAb products marketed in the United States reveals that the majority contain polysorbates.…”

Section: Discussionmentioning

confidence: 99%

Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration

Morar-Mitrica

Puri

Sassi

et al. 2015

mAbs

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The physical and chemical integrity of a biopharmaceutical must be maintained not only during long-term storage but also during administration. Specifically for the intravenous (i.v.) delivery of a protein drug, loss of stability can occur when the protein formulation is compounded with i.v. bag diluents, thus modifying the original composition of the drug product. Here we present the challenges associated with the delivery of a low-dose, highly potent monoclonal antibody (mAb) via the i.v. route. Through parallel in-use stability studies and conventional formulation development, a drug product was developed in which adsorptive losses and critical oxidative degradation pathways were effectively controlled. This development approach enabled the i.v. administration of clinical doses in the range of 0.1 to 0.5 mg total protein, while ensuring liquid drug product storage stability under refrigerated conditions.

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Induction and analysis of aggregates in a liquid IgG1-antibody formulation

Cited by 269 publications

References 35 publications

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration

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