Stability assessment of anti-bacterial antibodies in immunoglobulin G-depleted serum with validated immunoassays.

Aim: To investigate the stability of the anti-pneumococcal (PCP) and anti-haemophilus type B (Hib) immunoglobulins (IgGs) in human IgG-depleted serum samples frozen at -20°C. Materials & methods: Modified commercially available immunoassays (ELISAs) were bioanalytically validated. These ELISAs were used to measure levels of the two anti-bacterial IgG in samples kept at -20°C for up to 15 months. Human IgG-depleted serum was spiked with GAMMAGARD Liquid to obtain those samples. Results: Both ELISAs passed the validation test. Anti-PCP IgG and anti-Hib IgG were shown to be stable for at least 15 months at -20°C. Conclusion: These data confirm the stability of anti-bacterial IgG in human IgG-depleted serum and support the common practice of testing frozen samples.

Immunodeficiency disorders can prevent your body from fighting infections. These disorders make it easier to catch viruses and bacterial infections caused by so-called pathogens. Patients suffering from immunodeficiencies are treated throughout their lives with antibodies purified from human plasma. This immunoglobulin replacement therapy, which helps to avoid infections, provides specific antibodies directed against these pathogens. An antibody is a protein produced by the body's immune system to detect (bind) antigens and to help eliminating harmful substances. Little is known about the stability of such specific antibodies in samples taken from patients during clinical studies carried out to improve the replacement therapy. We investigated the stability of two such antibodies using a standard technique for their measurement. In a process termed validation, these methods were demonstrated to deliver accurate and precise results. For the stability study, we prepared human serum (= the liquid part of human blood) samples with specific antibodies levels expected in samples from patients on replacement therapy. These samples were kept frozen at -20°C for up to 15 months. The data obtained on analysis of the frozen samples showed the adequate stability of both antibodies directed against important pathogen. This stability confirms a common testing practice applied for samples obtained in clinical studies where usually such samples are not tested immediately but are stored frozen and tested in batches. In particular, the data for the two anti-bacterial antibodies support the storage of such samples for at least 15 months at -20°C before testing.

Selective and sensitive measurement of human neutrophil elastase in clinical samples based on a novel assay principle for protease activity measurement.

Imbalances between proteases and protease inhibitors have been associated with several pathological conditions including emphysema as seen in α1-antitrypsin deficiency. For this pathological condition, unimpeded neutrophil elastase activity has been ascribed a pivotal role in the destruction of lung tissue and thus in disease progression. Therefore, low, or non-quantifiable neutrophil elastase (NE) activity levels determined in bronchoalveolar lavage solutions indicate the success of α1-antitrypsin (AAT) augmentation therapy as NE activity will be erased. To overcome the known limitations of available elastase activity assays regarding sensitivity and selectivity, we developed a new elastase activity assay, which fundamentally relies on the highly specific complex formation between AAT and active elastase. Plate-bound AAT captured active elastase from the sample undergoing complex formation, followed by the immunological detection of human NE. This assay principle facilitated the measurement of low pM amounts of active human NE. The data of the assay performance check demonstrated adequate accuracy and precision profiles meeting currently accepted best practices for this activity assay, which can be classified as a ligand-binding assay. Furthermore, spike-recovery studies at low human NE levels, carried out for three human bronchoalveolar samples, resulted in recoveries within the 100 ± 20% range, while good linearity and parallelism of the samples' dilution-response curves was observed. Altogether, complemented by the data of selectivity and robustness studies and the accuracy and precision profile obtained in buffer, this newly developed human NE activity assay was demonstrated to perform accurately and precisely in clinically relevant samples.

Selective human factor VIII activity measurement after analytical in-line purification.

Background: It is essential to measure the activity of factor VIII (FVIII) throughout the life cycle of a coagulation FVIII concentrate. Such measurement in nonclinical pharmacokinetic studies is potentially biased by the presence of endogenous nonhuman FVIII, and certain manufacturing process-related additives can also impact the assay performance. Finally, the presence of FVIII activity-mimicking antibodies poses challenges when measuring FVIII in samples. Therefore, we developed an antibody-based chromogenic FVIII assay, which facilitates the selective and sensitive activity measurement of human FVIII in the presence of animal plasma and interfering agents. Methods: Plate-bound monoclonal anti-FVIII antibody specifically captured human FVIII, which was then measured with a chromogenic activity assay. A human reference plasma preparation was used to construct the calibration curve. Spike recovery was carried out in a citrated cynomolgus monkey plasma-solvent/detergent mixture and in the presence of the bispecific antibody emicizumab. Results: The calibration curve ranged from 3.03 to 97.0 mIU FVIII/ml and was obtained repeatedly with good accuracy. B domain-deleted and full-length FVIII did not differ in their responses. Recovery of spiked human FVIII in citrated cynomolgus monkey plasma was 102.7%, while neither native monkey plasma nor the other animal specimen tested showed any activity. Solvent/detergent solution and the bispecific antibody emicizumab had no influence on the assay. Conclusion: Combining antibody-mediated specific capture of human FVIII and a chromogenic activity assay resulted in a selective and sensitive measurement of human FVIII with no interference by endogenous, nonhuman FVIII, manufacturing process additives, or an FVIII activity-mimicking antibody.

Sensitive and specific measurement of alpha1-antitrypsin activity with an elastase complex formation immunosorbent assay (ECFISA).

Functionally active alpha1-antitrypsin (AAT) is measured predominantly with a chromogenic elastase inhibition assay, where the concentration of AAT activity inversely correlates with the levels of residual elastase. This standard assay has moderate sensitivity as it hardly allows the measurement of samples containing less than 10 µg of functionally active AAT per mL. To overcome this drawback, we developed a new assay format for the measurement of functionally active AAT, which we termed the elastase complex formation immunosorbent assay (ECFISA). The ECFISA uses plate-bound, still proteolytically active elastase, which attacks functionally active AAT under irreversible formation of a stable stochiometric 1 + 1 complex. This complex is then detected and measured by an anti-AAT peroxidase conjugate. Using three different approaches for the preparation of functionally inactive AAT - heating, oxidation, and complex formation with elastase - we confirmed beyond doubt that the ECFISA exclusively measures functionally active AAT and that these measurements are unimpaired by the presence of high concentrations of functionally inactive AAT. Studies addressing the coating procedure demonstrated that adequate and robust conditions had been defined for this essential first step of the ECFISA. Possible interference caused by the presence of important plasma proteinase inhibitors in the test samples could be excluded for the most abundant inhibitors. Even a 1.5-times molar excess of alpha2-macroglobulin over AAT was shown to have no impact, which is not the case for a conventional chromogenic activity assay. Functional activities determined with the ECFISA and validated chromogenic elastase inhibition assay matched well with a mean absolute bias of 0.64% calculated for the 25 samples measured. The results of the bioanalytical assay validation complied with the acceptance criteria for ligand-binding assays as given by current guidelines on validation of bioanalytical methods. Overall, the data obtained demonstrated the ECFISA as an accurate, precise, selective, and very sensitive method for AAT activity measurement at low levels previously inaccessible for direct measurement.

Polysialic Acid-Mediated Activity Measurement of Polysialylated Recombinant Coagulation Factor VIII.

Measurement of modified biologic including coagulation factors with extended half-life obtained, for example, by polysialylation pose an analytical challenge especially if both biological activity and presence of modification have to be determined. Analytical methods applied so far address only 1 of the 2 quality attributes of modified biologics. Here, we describe the development and bioanalytical validation of a polysialic acid-mediated factor VIII activity assay: Polysialic acid-specific capture of polysialylated recombinant factor VIII is combined with a chromogenic FVIII activity test using commercially available reagents. This assay principle enabled measurement of FVIII activity down to the pico mole-range without any interference by nonmodified factor VIII. To the best of our knowledge, this is the first method to selectively, accurately, and precisely measure simultaneously activity and modification integrity of a polysialylated biologic in complex matrices, as shown by the bioanalytical validation data. The convenience, robustness, and reliability of using this method has been demonstrated by its application for the nonclinical development of the polysialylated recombinant FVIII preparation. The method principle could be applied to protein modifications other than polysialylation and to activity tests other than the chromogenic FVIII assay.

Development of Methods for the Selective Measurement of the Single Amino Acid Exchange Variant Coagulation Factor IX Padua.

The description of hyper-functional factor IX (FIX) Padua triggered the development of BAX 335, an AAV8-based hemophilia B gene therapy vector designed to compensate for low FIX protein expression levels by expressing the FIX Padua variant, thereby reducing the exposure to viral vector. The presence of inactive FIX protein at baseline hindered conventional FIX:Ag ELISA from contributing to a more profound understanding of clinical data from the BAX 335 Phase 1/2 study (ClinicalTrials.gov: NCT01687608). By applying phage display technology, a Fab2 mini-antibody selectively binding to FIX Padua was developed and used to establish a FIX Padua-specific ELISA. The assay adequately performed, utilizing human and monkey plasma samples, and enabled the selective quantification of FIX Padua protein in human plasma samples from the BAX 335 trial. The mini-antibody also allowed the development of a chromogenic FIX Padua-specific activity assay, which adequately performed in human and mouse plasma. Collectively, the isolated FIX Padua-specific mini-antibody enabled the development of transgene-product-specific assays, which should improve the monitoring of hemophilia B gene therapies. The approach applied here for FIX Padua could be leveraged to develop variant-specific activity assays for other therapies where highly active enzymes are instrumental in achieving therapeutic levels of the transgene product.

Alkaline hydrolysis to increase the selectivity of colorimetric determination of polysorbate.

Here, we describe a straightforward sample pretreatment step for the colorimetric cobaltthiocyanate determination of polysorbate, which circumvents the assay's shortcomings due to interference of protein and does not require complex instrumentation. Protein-containing test samples are hydrolyzed with strong alkali at 100 °C, neutralized and clarified by filtration before applying the colorimetric assay. The modified method performs with appropriate accuracy and precision, allowing specific polysorbate measurement in the presence of Triton X-100 during virus inactivation, determination of residual amounts of polysorbate in the final products and measurement of polysorbate 80 in final formulated products. The alkaline hydrolysis step, primarily designed to provide the assay's reliability in the presence of protein, also enhances its selectivity towards interference by the non-ionic detergent Triton X-100 and increases its robustness against changes in the fatty acid moiety of polysorbate as it released the fatty acid essentially contributing to the known heterogeneity of polysorbates. These results demonstrate that with sample pretreatment the handy colorimetric assay, not requiring complex instrumentation, can be used to measure polysorbate 80 concentrations in intermediates and final products of therapeutic protein solutions.

Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.

BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays.

Development, Validation, and Application of a Novel Ligand-Binding Assay to Selectively Measure PEGylated Recombinant Human Coagulation Factor VIII (BAX 855).

BAX 855 is a PEGylated recombinant factor VIII preparation that showed prolonged circulatory half-life in nonclinical and clinical studies. This paper describes the development, validation, and application of a novel ligand-binding assay (LBA) to selectively measure BAX 855 in plasma. The LBA is based on PEG-specific capture of BAX 855, followed by immunological factor VIII (FVIII)-specific detection of the antibody-bound BAX 855. This assay principle enabled sensitive measurement of BAX 855 down to the low nanomolar range without interference from non-PEGylated FVIII as demonstrated by validation data for plasma from animals typically used for nonclinical characterization of FVIII. The selectivity of an in-house-developed anti-PEG and a commercially available preparation, shown by competition studies to primarily target the terminating methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, this new LBA adds to the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. The feasibility and convenience of using this method was demonstrated during extensive nonclinical characterization of BAX 855.

Convenient high-resolution isoelectric focusing (IEF) method for the separation of alpha1-proteinase inhibitor (A1PI) isoforms in A1PI concentrates.

Currently, high-resolution separation of A1PI is done in highly specialized laboratories using gels made in-house. This paper presents a high-resolution method for the separation of A1PI concentrates and human plasma using commercially available gels. Hybrid IEF was performed with carrier ampholytes and the gels were stained with Coomassie Brilliant Blue G-250. In addition, a sensitive immunoblotting procedure is described. The IEF method allowed the reproducible and convenient determination of the IEF pattern of A1PI in concentrates including resolution of glycan-dependent isoforms and isoproteins with secondary modifications such a C-terminal Lys-truncation. Furthermore, a shift in the IEF pattern of A1PI occurring upon reduction could be detected. Finally, in combination with a sample pretreatment step, the method proved able to monitor complex A1PI isoform patterns in samples with low A1PI concentrations as present for example in bronchoalveolar lavage solutions.