Compendial Methods: Suitability Verification, Challenges and Recommendations for Proteins.

Compendial testing methods are not required to be fully validated, but their suitability for testing should be verified under actual conditions of use. This requirement is established in 21 CFR 211.194(a)(2) of the current Good Manufacturing Practice regulations in the United States. ANVISA (Agência Nacional de Vigilância Sanitária) also requires that compendial analytical methods shall have their suitability demonstrated for the intended use by a partial validation study. Suitability verifications or partial validation can be divided into two major categories: visual and instrumental methods. For visual methods, the color and opalescence of interferences should be checked. If the color or clarity/opalescence of the sample is outside of the range of the Pharmacopeia standards/reference solutions, the validity of the test results should be evaluated. Specificity is usually waived because the methods are not specific to products, and accuracy/precision can be addressed by comparing results from analyst to analyst. For instrument methods, specificity can also be waived for certain assays. Accuracy is addressed by implementation of instrument calibration and/or method control. Precision is required either in suitability verification or when testing the samples. Here, we present approaches for suitability verification and the scientific rationale supporting compendial methods: visible particulates, subvisible particles, pH, osmolality, color and clarity/opalescence. Current challenges and recommendations are also discussed specifically for the analysis of protein products.

Photodisruption of the Structurally Conserved Cys-Cys-Trp Triads Leads to Reduction-Resistant Scrambled Intrachain Disulfides in an IgG1 Monoclonal Antibody.

Photostability conditions as prescribed by ICH guidelines induced highly reduction-resistant scrambled disulfides that contribute to the population of apparent nonreducible aggregates in an IgG1 mAb. Photoinduced cross-linked species were isolated under reducing conditions using an organic phase size exclusion chromatography (OP-SEC) method, followed by O18-labeling tryptic mapping to identify cross-linked peptides. Disulfide scrambling was observed within the IgG1 structurally conserved-intrachain cysteine-cysteine-tryptophan triads (Cys-Cys-Trp), and correlated with Trp-to-kynurenine (Kyn) photodegradation within these triads. We hypothesize that intrachain disulfides protect the proximal Trp within the Cys-Cys-Trp triads from photodegradation by enabling dissipation of Trp-absorbed UV energy via electron transfer to the disulfide bond. Finally, we propose three distinct mechanisms of photochemical degradation of monoclonal antibodies mediated by Trp residues.

Antitherapeutic antibody-mediated hepatotoxicity of recombinant human Apo2L/TRAIL in the cynomolgus monkey.

Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apoptosis. Recombinant human (rhu) Apo2L/TRAIL has been attractive as a potential cancer therapeutic because many types of tumor cells are sensitive to its apoptosis-inducing effects. Nonclinical toxicology studies were conducted to evaluate the safety of rhuApo2L/TRAIL for possible use in humans. The cynomolgus monkey was chosen for this safety assessment based on high protein sequence homology between human and cynomolgus Apo2L/TRAIL and comparable expression of their receptors. Although hepatotoxicity was observed in repeat-dose monkey studies with rhuApo2L/TRAIL, all animals that displayed hepatotoxicity had developed antitherapeutic antibodies (ATAs). The cynomolgus ATAs augmented the cytotoxicity of rhuApo2L/TRAIL but not of its cynomolgus counterpart. Of note, human and cynomolgus Apo2L/TRAIL differ by four amino acids, three of which are surface-exposed. In vivo studies comparing human and cynomolgus Apo2L/TRAIL supported the conclusion that these distinct amino acids served as epitopes for cross-species ATAs, capable of crosslinking rhuApo2L/TRAIL and thus triggering hepatocyte apoptosis. We describe a hapten-independent mechanism of immune-mediated, drug-related hepatotoxicity - in this case - associated with the administration of a human recombinant protein in monkeys. The elucidation of this mechanism enabled successful transition of rhuApo2L/TRAIL into human clinical trials.

A Spectral Method for Color Quantitation of a Protein Drug Solution.

Color is an important quality attribute for biotherapeutics. In the biotechnology industry, a visual method is most commonly utilized for color characterization of liquid drug protein solutions. The color testing method is used for both batch release and on stability testing for quality control. Using that method, an analyst visually determines the color of the sample by choosing the closest matching European Pharmacopeia reference color solution. The requirement to judge the best match makes it a subjective method. Furthermore, the visual method does not capture data on hue or chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we describe a quantitative method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. Following color industry standards established by International Commission on Illumination, this method converts a protein solution's visible absorption spectra to L*a*b* color space. Color matching is achieved within the L*a*b* color space, a practice that is already widely used in other industries. The work performed here is to facilitate the adoption and transition for the traditional visual assessment method to a quantitative spectral method. We describe here the algorithm used such that the quantitative spectral method correlates with the currently used visual method. In addition, we provide the L*a*b* values for the European Pharmacopeia reference color solutions required for the quantitative method. We have determined these L*a*b* values by gravimetrically preparing and measuring multiple lots of the reference color solutions. We demonstrate that the visual assessment and the quantitative spectral method are comparable using both low- and high-concentration antibody solutions and solutions with varying turbidity. LAY ABSTRACT: In the biotechnology industry, a visual assessment is the most commonly used method for color characterization, batch release, and stability testing of liquid protein drug solutions. Using this method, an analyst visually determines the color of the sample by choosing the closest match to a standard color series. This visual method can be subjective because it requires an analyst to make a judgment of the best match of color of the sample to the standard color series, and it does not capture data on hue and chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we developed a quantitative spectral method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. The details of the spectral quantitative method are described. A comparison between the visual assessment method and spectral quantitative method is presented. This study supports the transition to a quantitative spectral method from the visual assessment method for quality testing of protein solutions.

Validation of a Spectral Method for Quantitative Measurement of Color in Protein Drug Solutions.

A quantitative spectral method has been developed to precisely measure the color of protein solutions. In this method, a spectrophotometer is utilized for capturing the visible absorption spectrum of a protein solution, which can then be converted to color values (L*a*b*) that represent human perception of color in a quantitative three-dimensional space. These quantitative values (L*a*b*) allow for calculating the best match of a sample's color to a European Pharmacopoeia reference color solution. In order to qualify this instrument and assay for use in clinical quality control, a technical assessment was conducted to evaluate the assay suitability and precision. Setting acceptance criteria for this study required development and implementation of a unique statistical method for assessing precision in 3-dimensional space. Different instruments, cuvettes, protein solutions, and analysts were compared in this study. The instrument accuracy, repeatability, and assay precision were determined. The instrument and assay are found suitable for use in assessing color of drug substances and drug products and is comparable to the current European Pharmacopoeia visual assessment method. LAY ABSTRACT: In the biotechnology industry, a visual assessment is the most commonly used method for color characterization, batch release, and stability testing of liquid protein drug solutions. Using this method, an analyst visually determines the color of the sample by choosing the closest match to a standard color series. This visual method can be subjective because it requires an analyst to make a judgment of the best match of color of the sample to the standard color series, and it does not capture data on hue and chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we developed a quantitative spectral method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. In this study, we established a statistical method for assessing precision in 3-dimensional space and demonstrated that the quantitative spectral method is comparable with respect to precision and accuracy to the current European Pharmacopoeia visual assessment method.

Effect of ambient light on IgG1 monoclonal antibodies during drug product processing and development.

Photostability studies are standard stress testing conducted during drug product development of various pharmaceutical compounds, including small molecules and proteins. These studies as recommended by ICH Q1B are carried out using no less than 1.2× 10(6)lux-hours in the visible region and no less than 200Wh/m(2) in UV light. However, normal drug product processing is carried out under fluorescent lamps that emit white light almost exclusively in the >400nm region with a small UV quotient. We term these as ambient or mild light conditions. We tested several IgG1 monoclonal antibodies (mAbs 1-5) under these ambient light conditions and compared them to the ICH light conditions. All the mAbs were significantly degraded under the ICH light but several mAbs (mAbs 3-5) were processed without impacting any product quality attributes under ambient or mild light conditions. Interestingly we observed site-specific Trp oxidation in mAb1, while higher aggregation and color change were observed for mAb2 under mild light conditions. The recommended ICH light conditions have a high UV component and hence may not help to rank order photosensitivity under normal protein DP processing conditions.

In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability.

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery.

Probing antibody internal dynamics with fluorescence anisotropy and molecular dynamics simulations.

The solution dynamics of antibodies are critical to antibody function. We explore the internal solution dynamics of antibody molecules through the combination of time-resolved fluorescence anisotropy experiments on IgG1 with more than two microseconds of all-atom molecular dynamics (MD) simulations in explicit water, an order of magnitude more than in previous simulations. We analyze the correlated motions with a mutual information entropy quantity, and examine state transition rates in a Markov-state model, to give coarse-grained descriptors of the motions. Our MD simulations show that while there are many strongly correlated motions, antibodies are highly flexible, with F(ab) and F(c) domains constantly forming and breaking contacts, both polar and non-polar. We find that salt bridges break and reform, and not always with the same partners. While the MD simulations in explicit water give the right time scales for the motions, the simulated motions are about 3-fold faster than the experiments. Overall, the picture that emerges is that antibodies do not simply fluctuate around a single state of atomic contacts. Rather, in these large molecules, different atoms come in contact during different motions.

Stability of IgG1 monoclonal antibodies in intravenous infusion bags under clinical in-use conditions.

Compounding pharmacists are expected to prepare safe and efficacious doses of medication under time and economical constraints while protecting pharmacy staff and caregivers from inadvertent exposure to the drug. The pharmacist has the additional responsibility to ensure that the product is stable in the final-administrated form as the time between drug preparation and administration is considerable. Pharmacists are responsible for setting a "beyond-use" date based on United States Pharmacopeia 797, wherein the beyond-use date for the compounded sterile preparation (CSP) is defined as the time by which the compounded preparation must be used to avoid risks for product degradation, contamination, and so on. Physical and chemical stability of the CSP can be difficult to maintain over extended storage, especially since the formulation components are diluted within the intravenous (i.v.) bag contents. Recent published reports have suggested the use of extended time, beyond that recommended by the manufacturer, for the storage and administration of CSP. These recommendations were based on inadequate analytical testing of the CSP. Herein, we demonstrate that setting of the beyond-use date should be carefully assessed using the appropriate analytical methods and testing. Results from our studies clearly indicate that many of the tested IgG(1) monoclonal antibodies should not be diluted and stored in i.v. bags over extended period of time, and particularly should not be transported after dilution in the infusion bags without consulting the manufacturer. Results from this study also indicate that i.v. bag agitation studies should be performed during pharmaceutical development of protein therapeutics under clinical in-use conditions, especially when storage and transportation of i.v. bags are possible in global clinical trials and post-licensure usage.

Application of frequency-modulated spectroscopy in vacuum seal integrity testing of lyophilized biological products.

This study examined the feasibility of applying frequency-modulated spectroscopy (FMS) to test vacuum seal integrity of lyophilized protein pharmaceuticals in glass vials. A lyophilized recombinant monoclonal antibody was used as an example to demonstrate that FMS is a non-destructive method that could accurately and quickly determine vial vacuum integrity within a pressure range of 0.04 to 0.5 atm. The coefficient of determination (R2) of a bench-top instrument was found to be >0.99. Only seconds were required to analyze each sample. The instrument sensitivity and specificity were 0.95 and >0.99, respectively, based on analysis of approximately 40,000 samples. Because of low energy input by the instrument, no adverse effect on the protein quality was found immediately after up to 1 h of continuous laser exposure. The laser-exposed samples had comparable stability to non-exposed control vials after 12 weeks of storage at 40 degrees C.