A high-resolution multi-attribute method for product characterization, process characterization, and quality control of therapeutic proteins.

During the manufacturing of therapeutic proteins, Critical Quality Attributes (CQAs) have been monitored by conventional methods, such as cation exchange chromatography (CEX), reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS), and 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelling method. The conventional methods often generate individual peaks that contain multiple components, which may obscure the detection and the quantification of individual critical quality attributes (CQAs). Alternatively, Multi-Attribute Method (MAM) enables detection and quantification of specific CQAs. A high resolution MAM has been developed and qualified to replace several conventional methods in monitoring product quality attributes, such as oxidation, deamidation, clipping, and glycosylation. The qualified MAM was implemented in process characterization, as well as release and stability assays in quality control (QC). In combination with a design-of-experiments (DoE), the MAM method identified multivariate process parameter ranges that yield acceptable CQA level, which provides operational flexibility for manufacturing.

Identification of critical chemical modifications and paratope mapping by size exclusion chromatography of stressed antibody-target complexes.

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding.

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis.

Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.

Comparison of Acid Titration, Conductivity, Flame Photometry, ICP-MS, and Accelerated Lamellae Formation Techniques in Determining Glass Vial Quality.

Certain types of glass vials used as primary containers for liquid formulations of biopharmaceutical drug products have been observed with delamination that produced small glass like flakes termed lamellae under certain conditions during storage. The cause of this delamination is in part related to the glass surface defects, which renders the vials susceptible to flaking, and lamellae are formed during the high-temperature melting and annealing used for vial fabrication and shaping. The current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. Four alternative techniques with improved throughput, convenience, and/or comprehension were examined by subjecting seven lots of vials to analysis by all techniques. The first three new techniques of conductivity, flame photometry, and inductively coupled plasma mass spectrometry measured the same sample pools as acid titration. All three showed good correlation with alkalinity: conductivity (R(2) = 0.9951), flame photometry sodium (R(2) = 0.9895), and several elements by inductively coupled plasma mass spectrometry [(sodium (R(2) = 0.9869), boron (R(2) = 0.9796), silicon (R(2) = 0.9426), total (R(2) = 0.9639)]. The fourth technique processed the vials under conditions that promote delamination, termed accelerated lamellae formation, and then inspected those vials visually for lamellae. The visual inspection results without the lot with different processing condition correlated well with alkalinity (R(2) = 0.9474). Due to vial processing differences affecting alkalinity measurements and delamination propensity differently, the ratio of silicon and sodium measurements from inductively coupled plasma mass spectrometry was the most informative technique to assess overall vial quality and vial propensity for lamellae formation. The other techniques of conductivity, flame photometry, and accelerated lamellae formation condition may still be suitable for routine screening of vial lots produced under consistent processes. LAY ABSTRACT: Recently, delamination that produced small glass like flakes termed lamellae has been observed in glass vials that are commonly used as primary containers for pharmaceutical drug products under certain conditions during storage. The main cause of these lamellae was the quality of the glass itself related to the manufacturing process. Current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. As alternative to the European Pharmacopoeia method, four other techniques were assessed. Three new techniques of conductivity, flame photometry, and inductively coupled plasma mass spectrometry measured the vial extract pool as acid titration to quantify quality, and they demonstrated good correlation with original alkalinity. The fourth technique processed the vials under conditions that promote delamination, termed accelerated lamellae formation, and the vials were then inspected visually for lamellae. The accelerated lamellae formation technique also showed good correlation with alkalinity. Of the new four techniques, inductively coupled plasma mass spectrometry was the most informative technique to assess overall vial quality even with differences in processing between vial lots. Other three techniques were still suitable for routine screening of vial lots produced under consistent processes.

Do not drop: mechanical shock in vials causes cavitation, protein aggregation, and particle formation.

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 µs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond time course. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size-exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues.

A molecular mechanism of hydrolysis of peptide bonds at neutral pH using a model compound.

The covalent stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. In this computational study, the hydrolytic reaction of peptide bonds at neutral pH was studied using a model compound, N-MAA. The most probable reaction pathway and intermediate(s) involved are controversial in previous studies. In addition, most previous computational studies focus on the energetics of chemical species involved, rather than providing a dynamic picture of the reaction process in aqueous conditions. However, fluctuations at finite temperatures are quite important, as we show. Thus, a path sampling method was used to generate an ensemble of trajectories according to their statistical weights in trajectory space. An ab initio molecular dynamics technique was applied to advance the time of the reaction in order to collect trajectories. The likelihood maximization procedure and its modification were used in extracting dynamically relevant degrees of freedom in the system, and approximations of the reaction coordinate were compared. It was found that this hydrolytic reaction is very complex because it involves many degrees of freedom. The reaction coordinate C-O distance previously assumed was found to be inadequate in describing the dynamic progress of the reaction. In addition to affecting atoms directly involved in bond-making and -breaking processes, the water network also has determining effects on the hydrolytic reaction, a fact which is manifest in the expression of the best one-dimensional reaction coordinate that we found, which includes five geometric quantities. p(B) histograms were computed to verify the results of the likelihood maximization and to evaluate the accuracy of our best reaction coordinate to the "true" reaction coordinate. The relation with previous suggested reaction pathways and intermediate(s) is discussed in terms of computational system, method, and accuracy.

Molecular mechanism of acid-catalyzed hydrolysis of peptide bonds using a model compound.

The stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. Recent studies found elevated nonenzymatic hydrolysis in the hinge region of antibody molecules, but no mechanism was identified. As a first step in providing a mechanistic interpretation, this computational study examines the rate-determining step of the hydrolytic reaction of a peptide bond under acidic pH by a path sampling technique using a model compound N-MAA. Most previous computational studies did not include explicit water molecules, whose effects are significant in solution chemistry, nor did they provide a dynamic picture for the reaction process in aqueous conditions. Because no single trajectory can be used to describe the reaction dynamics due to fluctuations at finite temperatures, a variant version of the transition path sampling technique, the aimless shooting algorithm, was used to sample dynamic trajectories and to generate an ensemble of transition trajectories according to their statistical weights in the trajectory space. Each trajectory was computed as the time evolution of the molecular system using the Car-Parrinello molecular dynamics technique. The likelihood maximization procedure and its modification were used in extracting dynamically relevant degrees of freedom in the system, and approximations of the reaction coordinate were compared. Its low log-likelihood score and poor p(B) histogram indicate that the C-O distance previously assumed as the reaction coordinate for the rate-determining step is inadequate in describing the dynamics of the reaction. More than one order parameter in a candidate set including millions of geometric quantities was required to produce a convergent reaction coordinate model; its involvement of many degrees of freedom suggests that this hydrolytic reaction step is very complex. In addition to affecting atoms directly involved in bond-making and -breaking processes, the water network also has determining effects on the hydrolytic reaction, a fact that is manifest in the expression of the one-dimensional best-ranked reaction coordinate, which includes three geometric quantities. The p(B) histograms were computed to verify the results of the likelihood maximization and to verify the accuracy of approximation to the "true" reaction coordinate.

Conformational implications of an inversed pH-dependent antibody aggregation.

Antibody formulation development relies on accelerated stability data at elevated temperatures to optimize formulation parameters. However, the pH- and temperature-dependence of aggregation is complicated for antibody formulations. In this study, a human monoclonal IgG2 antibody exhibited typical pH-dependent dimer formation under normal storage conditions (4 and/or 29 degrees C). However, an inversed pH-dependence was discovered for high molecular weight aggregate formation at elevated temperatures (37 degrees C). The different stability profiles exhibited at the various storage conditions resulted in nonlinearity of the Arrhenius kinetics. Thermal unfolding at or below 37 degrees C was not evident by differential scanning calorimetry. Enriched populations of the structural isoforms of the IgG2 subclass were tested for their unique temperature and pH-dependence of aggregation. The Arrhenius kinetics of aggregation for each of the individual IgG2 isoforms was also nonlinear. However, the temperature-dependence of clipping suggested that clip-mediated aggregation was responsible for the increased higher order aggregates at low pH and elevated temperatures. Unique clip species resulting from the conformational differences between the IgG2 isoforms lead to increased aggregation. These results have implications on the mechanisms of antibody aggregation and on the validity of accelerated data to predict shelf-life accurately.

Comparative oxidation studies of methionine residues reflect a structural effect on chemical kinetics in rhG-CSF.

The effect of protein conformation on the rate of chemical degradation is poorly understood. To address the role of structure on chemical degradation kinetics, comparative oxidation studies of methionine residues in recombinant human granulocyte colony-stimulating factor (rhG-CSF) were performed. The kinetics of oxidation of methionine residues by hydrogen peroxide (H2O2) in rhG-CSF and corresponding chemically synthesized peptides thereof was measured at different temperatures. To assess structural effects, equilibrium denaturation experiments also were conducted on rhG-CSF, yielding the free energy of unfolding as a function of temperature. A comparison of the relative rates of oxidation of methionine residues in short peptides with those of corresponding methionine residues in rhG-CSF yields an understanding of how protein tertiary structure affects oxidation reactions. For the temperature range that was studied, 4-45 degrees C, the oxidation rate constants followed an Arrhenius equation quite well, suggesting the lack of temperature-induced local structural perturbations that affect chemical degradation rates. One of the four methionine residues, Met 122, exhibited an activation energy significantly different from that of the corresponding peptide. Extrapolation of kinetic data predicts non-Arrhenius behavior around the melting temperature. Three phenomenological models based on different mechanisms are discussed, and an application to shelf life prediction of pharmaceuticals is presented.