Richard R. Rustandi

Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies

Within pharmaceutical industry charge heterogeneity testing of biopharmaceuticals has to be reproducible and fast. It should pass method validation according to ICH Q2. Classical approaches for the analysis of the charge heterogeneity of biopharmaceuticals are ion exchange chromatography (IEC) and isoelectric focusing (IEF). As an alternative approach, also capillary zone electrophoresis (CZE) was expected to allow reliable charge heterogeneity profiling by separation according to the analytes net charge and hydrodynamic radius. Aim of this study was to assess if CZE possesses all of the required features. Therefore, beside lab internal validation of this method also an international cross company study was organized. It was shown that CZE is applicable across a broad pI range between 7.4 and 9.5. The coefficient of correlation was above 0.99 which demonstrated linearity. Precision by repeatability was around 1% (maximum relative standard deviation per level) and accuracy by recovery was around 100% (mean recovery per level). Accuracy was further verified by direct comparison of IEC, IEF and CZE, which in this case showed comparable %CPA results for all three methods. However, best resolution for the investigated MAb was obtained with CZE. In dependence on sample concentration the detection limit was between 1 and 3%. Within the intercompany study for CZE the same stressed and non-stressed samples were analyzed in each of the 11 participating labs. The finally obtained dataset contained more than 1000 separations which provided an extended dataset for further statistical evaluation. Among the different labs no significant differences between the peak profiles were observed. Mean driver for dropouts in quantitative evaluation was linked to the performance of some participating labs while the impact of the method performance was negligible. In comparison to a 50cm capillary there was a slightly better separation of impurities and drug substance related compounds with a 30cm capillary which demonstrates that an increased stability indicating potential can be combined with the increased separation velocity and high throughput capability of a shorter capillary. Separation can be performed in as little as approx. 3min allowing high throughput applications. The intercompany study delivered precise results without explicit training of the participating labs in the method prior to the study (standard deviations in the range of 1%). It was demonstrated that CZE is an alternative platform technology for the charge heterogeneity testing of antibodies in the pharmaceutical industry.

Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex

ABSTRACT Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen—the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains. IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen—the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies.

Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis.

Application of Capillary Electrophoresis in Glycoprotein Analysis

Capillary electrophoresis (CE) is a versatile analytical method used to characterize glycoproteins. We have used several modes of CE separation such as CE-SDS gel, imaged capillary isoelectric focusing (icIEF), and capillary zone electrophoresis (CZE) to study therapeutic glycoprotein products. CE-SDS gel is applied to characterize the glycan occupancy and number of glycosylation sites, and icIEF is used to study the charge heterogeneities due to sialic acids in glycoproteins. To further characterize the glycoprotein, removal of N-linked glycans is necessary and a CZE technique is employed to analyze each glycan moiety. Examples from a monoclonal antibody, erythropoietin, and granulocyte colony-stimulating factor are presented here to demonstrate the utility of these CE modes. The details of sample preparation and separation conditions for each CE mode are described in this chapter.

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Toxicity assessment of Clostridium difficile toxins in rodent models and protection of vaccination.

Clostridium difficile is the leading cause of hospital-acquired diarrhea, also known as C. difficile associated diarrhea. The two major toxins, toxin A and toxin B are produced by most C. difficile bacteria, but some strains, such as BI/NAP1/027 isolates, produce a third toxin called binary toxin. The precise biological role of binary toxin is not clear but it has been shown to be a cytotoxin for Vero cells. We evaluated the toxicity of these toxins in mice and hamsters and found that binary toxin causes death in both animals similar to toxins A and B. Furthermore, immunization of mice with mutant toxoids of all three toxins provided protection upon challenge with native toxins. These results support the concept that binary toxin contributes to the pathogenicity of C. difficile and provide a method for monitoring the toxicity of binary toxin components in vaccines.

Complement enhances in vitro neutralizing potency of antibodies to human cytomegalovirus glycoprotein B (gB) and immune sera induced by gB/MF59 vaccination

Human cytomegalovirus (HCMV) is the leading cause of in utero viral infection in the United States. Since congenital HCMV infection can lead to birth defects in newborns, developing a prophylactic vaccine is a high priority. One of the early experimental vaccines, composed of a recombinant glycoprotein B (gB) formulated with MF59 adjuvant, has demonstrated approximately 50% efficacy against HCMV infection in seronegative women. Using immune sera from two gB/MF59 Phase 1 studies in humans we showed that complement can enhance the in vitro HCMV neutralizing potency of antibodies induced by the gB/MF59 vaccination. To characterize this complement-dependent antiviral activity, we analyzed three rabbit non-neutralizing gB monoclonal antibodies (mAbs) with different biochemical profiles including epitope specificity. Two of the three mAbs, r272.7 and r210.4, exhibited neutralizing activity when complement was added to the assays, and this complement-dependent antiviral activity was not related to the antibody’s affinity to gB but appeared to be associated with their epitope specificities. Moreover, neutralization could only be demonstrated when complement was present at or before viral entry, suggesting that IgG Fc-mediated function was not the basis for this antiviral activity. Lastly, we demonstrated that gB/MF59 immune sera contained antibodies that can cross-compete with r272.7 for gB binding and that the titers of these antibodies correlated with complement-dependent neutralization titers. These results suggested that gB antibodies with certain biochemical properties have neutralizing potency when complement is present and that this complement-dependent antiviral activity may be a part of immune components which conferred protection against HCMV infection by gB/MF59 vaccination.Fetal infection: Complementing vaccine performanceEnhancing a cytomegalovirus vaccine candidate with immune system proteins could be a step closer to protection against pre-birth infections. Cytomegalovirus is the leading cause of in utero infections in the United States; however, the leading vaccine candidate against the virus is, at best, 50% effective. A US group of scientists led by Merck’s Tong-Ming Fu found that adding a set of innate, antipathogenic proteins called ‘complement’ to the candidate increased the protective effects of immunization in humans, and in vitro with a selection of otherwise non-protective antibodies. The team also found that complement augmented the vaccine through a non-standard mechanism, which was dependent on the specific profile of the antibodies fighting the infection. This article describes an interesting mechanism of vaccine protection that warrants further investigation.

Hydrophobic Interaction Chromatography to Analyze Glycoproteins

Hydrophobic interaction chromatography (HIC) is one of many separation techniques that can be used to analyze proteins. The separation mechanism is based on the adsorption of the hydrophobic region of the protein to the hydrophobic ligands attached to the column in the presence of high salt. The proteins are then eluted by descending salt concentration. Here we describe the use of this HIC technique to evaluate the hydrophobicity of different monoclonal antibodies (mAbs) and to separate different heterogeneities that occur in mAb.

Quantitation of CRM197 using imaged capillary isoelectric focusing with fluorescence detection and capillary Western

Maurice is a new instrument that can perform imaged capillary isoelectric focusing (icIEF). The standard detection for icIEF is UV absorbance at 280xa0nm, which limits its application to high protein concentration samples and non-complex samples. Here we describe an icIEF instrument with fluorescence detection. We demonstrate the advantage of using either icIEF with fluorescence detection or quantitative Western Blot to measure diphtheria toxin mutant CRM197 protein titer in crude cell lysates and purified samples. These two techniques have great potentials to become standard methods to analyze protein titers in crude cell lysate or other complex samples types.

Ion-Exchange Chromatography to Analyze Components of a Clostridium difficile Vaccine

Ion-exchange (IEX) chromatography is one of many separation techniques that can be employed to analyze proteins. The separation mechanism is based on a reversible interaction between charged amino acids of a protein to the charged ligands attached to a column at a given pH. This interaction depends on both the pI and conformation of the protein being analyzed. The proteins are eluted by increasing the salt concentration or pH gradient. Here we describe the use of this technique to characterize the charge variant heterogeneities and to monitor stability of four protein antigen components of a Clostridium difficile vaccine. Furthermore, the IEX technique can be used to monitor reversion to toxicity for formaldehyde-treated Clostridium difficile toxins.