Flow virometry for process monitoring of live virus vaccines-lessons learned from ERVEBO.

Direct at line monitoring of live virus particles in commercial manufacturing of vaccines is challenging due to their small size. Detection of malformed or damaged virions with reduced potency is rate-limited by release potency assays with long turnaround times. Thus, preempting batch failures caused by out of specification potency results is almost impossible. Much needed are in-process tools that can monitor and detect compromised viral particles in live-virus vaccines (LVVs) manufacturing based on changes in their biophysical properties to provide timely measures to rectify process stresses leading to such damage. Using ERVEBO, MSD's Ebola virus vaccine as an example, here we describe a flow virometry assay that can quickly detect damaged virus particles and provide mechanistic insight into process parameters contributing to the damage. Furthermore, we describe a 24-h high throughput infectivity assay that can be used to correlate damaged particles directly to loss in viral infectivity (potency) in-process. Collectively, we provide a set of innovative tools to enable rapid process development, process monitoring, and control strategy implementation in large scale LVV manufacturing.

Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes.

Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128-131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128-131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)2 homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0-6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4-8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128-131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.

Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

Demonstrating ß-glucan and yeast peptide clearance in biopharmaceutical downstream processes.

The use of yeast- and plant-derived hydrolysates in cell culture production processes has sparked concerns over the potential immunogenicity risk posed by ß-glucans and yeast peptides contained in these raw materials. This article utilizes a combination of in-process testing from large-scale manufacturing and scale-down spiking studies to demonstrate the clearance of ß-glucans and yeast peptides through chromatographic steps in the downstream purification process for a monoclonal antibody. ß-Glucans were found to flow through most all three modes of chromatography (Protein A, cation and anion exchange) without binding to the resins or the product. Protein A affinity chromatography was found to provide the best clearance factor. The efficacy of the resin sanitization and storage procedures to prevent carryover from one run to the next was also demonstrated. Yeast peptides were found to be metabolized during the cell culture process and were undetectable after the Protein A purification step. The data presented here serve to allay concerns about the use of hydrolysates in cell culture production. The methodology presented here provides a template to demonstrate clearance of ß-glucans and yeast peptides through chromatographic steps in downstream processing.

A simple reversed phase high-performance liquid chromatography method for polysorbate 80 quantitation in monoclonal antibody drug products.

In this paper, we discuss an improved high-performance liquid chromatography (HPLC) method for the quantitation of polysorbate 80 (polyoxyethylenesorbitan monooleate), a commonly used stabilizing excipient in therapeutic drug solutions. This method is performed by quantitation of oleic acid, a hydrolysis product of polysorbate 80. Using base hydrolysis, polysorbate 80 releases the oleic acid at a 1:1 molar ratio. The oleic acid can then be separated from other polysorbate 80 hydrolysis products and matrices using reversed phase HPLC. The oleic acid is monitored without derivatization using the absorbance at 195 nm. The method was validated and also shown to be accurate for the quantitation of polysorbate 80 in a high protein concentration monoclonal antibody drug product. For the measured polysorbate 80 concentrations, the repeatability was less than 6.2% relative standard deviation of the mean (% RSD) with the day-to-day intermediate precision being less than 8.2% RSD. The accuracy of the oleic acid quantitation averaged 94-109% in different IgG(1) and IgG(4) drug solutions with variable polysorbate 80 concentrations. In this study, polyoxyethylene, a by-product of the polysorbate 80 hydrolysis was also identified. This peak was not identified by previous methods and also increased proportionally to the polysorbate 80 concentration. We have developed and qualified a method which uses common equipment found in most laboratories and is usable over a range of monoclonal antibody subclasses and protein concentrations.

Isomerization in the CDR2 of a monoclonal antibody: Binding analysis and factors that influence the isomerization rate.

Isomerization of a monoclonal antibody is one of the common routes of protein degradation. An isomerization in the complementarity-determining region (CDR) was found previously and is investigated in depth in this work. Affinity analysis proves that the antibody with one isomerized heavy chain has lower binding. Binding constants were determined, and exhibited a slower on-rate in conjunction with a faster off-rate for this isomerization. To determine the role of the buffer on the rate of isomerization, this antibody was incubated in various matrices and the amount of isomerized antibody was determined by hydrophobic interaction chromatography (HIC). The rate was found to be dependent on the pH as well as the net negative charge of the buffer components that can act as proton acceptors. An Arrhenius plot was performed to predict the levels of isomerization and a comparison of real samples proved the model was correct. This work affirms that isomerization in the CDR of a therapeutic antibody is important to monitor and the formulation buffer plays a significant role in the rate of the isomerization.

Identification and measurement of isoaspartic acid formation in the complementarity determining region of a fully human monoclonal antibody.

Isomerization plays a key role in protein degradation. This isomerization is often difficult to detect by many protein characterization methods such as SDS-PAGE, SEC, and IEF. This work shows the identification of an isomerized aspartic acid residue in the CDR2 of the heavy chain of a fully human monoclonal antibody. This isoaspartic acid increases significantly with storage at 2-8 degrees C. Hydrophobic interaction chromatography was utilized to separate the isoaspartic variant in the intact state. Mass spectrometry including peptide mapping was employed to identify and confirm the exact location of the modification. Since this modification occurs in the complementarity determining region (CDR) it was found that binding is reduced. Therefore, three different analytical methods for regular analysis of this isomerization are evaluated. These methods include peptide mapping by LC-MS, HIC, and a protein isoaspartate methyltransferase assay. It was determined that HIC is the best method to regularly assay the level of isomerization in this monoclonal antibody.

Investigation of proteins and peptides from yeastolate and subsequent impurity testing of drug product.

Hydrolysates play an important role in modern biological production. These mixtures are mostly undefined and contain a mixture of proteins, peptides, and amino acids along with other non-amino acid-based components. Recently, there has been an interest in defining and sequencing proteins and peptides in these hydrolysates to subsequently develop an assay to ensure removal during product purification. This work investigates an ultrafiltrate of yeastolate to determine whether any protein is present. Size exclusion chromatography indicated a possible high molecular weight component (>10 kDa). This suspected high molecular weight fraction was collected and investigated. It was determined that this fraction consists of nucleic acids; and no protein was detected using sensitive modern techniques including HPLC, mass spectrometry, and SDS-PAGE. Next, five unique, yeast-specific peptides were identified, sequenced, and confirmed. Finally, an impurity assay for any residual yeast specific peptides was developed and the analytical metrics were determined including accuracy, precision, linearity, range, and limits of detection and quantitation.

Evaluation of oligosaccharide methods for carbohydrate analysis in a fully human monoclonal antibody and comparison of the results to the monosaccharide composition determination by a novel calculation.

Carbohydrates can change a drug's properties including solubility, affinity towards antigen, pharmacokinetics and pharmacodynamics. Due to this importance, carbohydrate composition is utilized as a parameter to evaluate a drug candidate's quality. In this study, the compositional monosaccharides of a drug candidate are measured by HPAEC-PAD, while the oligosaccharides are studied by HPAEC-PAD, CE-LIF and LC-MS. The advantages and limitations of these various approaches for oligosaccharide analysis are reviewed in this work. While the methods used for oligosaccharide analysis are well established we have devised a new and novel calculation for determining monosaccharide content using the relative percentages of the N-glycans. This calculation was used to evaluate the accuracy of the oligosaccharide determination methods by comparison of the N-glycan data to the experimental monosaccharide data. The results obtained from this novel calculation demonstrate that the relative abundance of carbohydrates as determined from these various approaches are consistent.

Peptide mapping of therapeutic monoclonal antibodies: improvements for increased speed and fewer artifacts.

Peptide mapping is a widely utilized technique to characterize monoclonal antibodies for the purpose of product identity and is becoming increasing important as a stability indicating assay. Many conventional peptide-mapping methods are extremely time consuming and yield a map that is wrought with processing artifact peaks such as deamidation, carbamylation, and missed cleavages. Therefore, this work examines the many common individual sample preparation steps of the peptide-mapping procedure for monoclonal antibodies including the steps of denaturing, reduction, sample cleanup, digestion, and HPLC solvent selection. Improvements in each of these steps are demonstrated that greatly help to reduce artifacts and also allow for reduction of overall sample preparation time. After evaluating the many different parameters for increased speed and fewer artifacts, the sample preparation was reduced from days to hours and the resulting peptide map is nearly free of sample or background artifacts. Therefore, this peptide map procedure and optimization scheme is an excellent tool to further examine real sample changes in a shorter amount of time.

C-terminal lysine variants in fully human monoclonal antibodies: investigation of test methods and possible causes.

The C-terminal lysine variation is commonly observed in biopharmaceutical monoclonal antibodies. This modification can be important since it is found to be sensitive to the production process. The methods commonly used to probe this charge variation, including IEF, cIEF, ion-exchange chromatography, and LC-MS, were evaluated for their ability to effectively approximate relative percentages of lysine variants. A monoclonal antibody produced in a B cell hybridoma versus a CHO cell transfectoma was examined and it was determined that the relative amount of incorporated C-terminal lysine can vary greatly between these two production schemes. Another case study is shown whereby a different monoclonal antibody is subject to some minor process changes and the extent of lysine variation also exhibits a significant difference. During these studies the different methods for determining the extent of variation were evaluated and it was determined that LC-MS after trypsin digestion provides reproducible relative percentage information and has significant advantages over other methods. The final section of this work investigates the possible origins of this modification and evidence is shown that carboxypeptidase B or another basic carboxypeptidase causes this variation.

Affinity capture and detection of immunoglobulin E in human serum using an aptamer-modified surface in matrix-assisted laser desorption/ionization mass spectrometry.

Capture and detection of immunoglobulin E (IgE) in simple solution and in human serum using an aptamer-modified probe surface for affinity matrix-assisted laser desorption/ionization mass spectroscopy detection is reported. Detectable signals were obtained for 1 amol of IgE applied either in a single, 1microL application of 1 pM IgE or after 10 successive, 1-microL applications of 100 fM IgE. In both cases, the surface was rinsed after each application of IgE to remove sample concomitants including salts and free or nonspecifically associated proteins. Detection of native IgE, which is the least abundant of the serum immunoglobulins and occurs at subnanomolar levels, in human serum was demonstrated and interference from the high-abundance immunoglobulins and albumin was investigated. The aptamer-modified surface showed high selectivity toward immunoglobulins in serum, with no significant interference from serum albumin. Addition of IgE to the serum suppressed the signals from the other immunoglobulins, confirming the expected selectivity of the aptamer surface toward IgE. Dilution of the serum increased the selectivity toward IgE; the protein was detected without interference in a 10,000-fold dilution of the serum, which is consistent with detection of IgE at amol (pM) levels in standard solutions.

Determination of the origin of the N-terminal pyro-glutamate variation in monoclonal antibodies using model peptides.

The purpose of this work is to determine the cause of the cyclization of the N-terminal glutamine in recombinant proteins and monoclonal antibodies. This cyclization reaction commonly occurs on the N-terminal of light and/or heavy chains of antibodies and leads to heterogeneity of the final product. Two model peptides and an antibody containing an N-terminal glutamine were used to investigate the formation of N-terminal pyro- glutamic acid under various experimental conditions and different stages of the biosynthetic process. LC-MS analysis was used to separate and quantify the N-terminal variants. Experiments prove that the cyclization reaction is spontaneous and highly dependent on temperature and buffer composition and less dependent on pH. The conditions presented in most biopharmaceutical processes accelerate the formation of this variant. The majority of the near complete conversion (>95%) of N-terminal glutamine to pyro-glutamic acid commonly observed for antibodies appears to occur inside the bioreactor with only a small contribution from purification, formulation, and analytical preparation.

Aptamer-enhanced laser desorption/ionization for affinity mass spectrometry.

The thrombin-binding DNA aptamer was used for affinity capture of thrombin in MALDI-TOF-MS. The aptamer was covalently attached to the surface of a glass slide that served as the MALDI surface. Results show that thrombin is retained at the aptamer-modified surface while nonspecific proteins, such as albumin, are removed by rinsing with buffer. Upon application of the low-pH MALDI matrix, the G-quartet structure of the aptamer unfolds, releasing the captured thrombin. Following TOF-MS analysis, residual matrix and protein can be washed from the surface, and buffer can be applied to refold the aptamers, allowing the surface to be reused. Selective capture of thrombin from mixtures of thrombin and albumin and of thrombin and prothrombin from human plasma was demonstrated. This simple approach to affinity capture, isolation, and detection holds potential for analysis, sensing, purification, and preconcentration of proteins in biological fluids.