Establishing a control system using QbD principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the elements and tools used to establish acceptance criteria and an attribute testing strategy (ATS) for product variants and process related impurities. The acceptable ranges for CQAs are set based on their potential impact on efficacy and safety/immunogenicity. This approach is focused on the management of patient impacts, rather than simply maintaining a consistent analytical profile. The ATS tools were designed to identify quality attributes that required process and/or testing controls, or that could be captured in a monitoring system to enable lifecycle management of the control strategy.

Structure and function of purified monoclonal antibody dimers induced by different stress conditions.

PURPOSE: To investigate structure and function of different monoclonal antibody (MAb) dimers. METHODS: MAb dimers were induced by process-related, low pH and UV light stress. Dimers were isolated and purified by chromatography and extensively characterized by biochemical, structural and functional methods. RESULTS: Highly purified dimer forms were obtained which enabled detailed characterization. Dimers induced by process stress were associated by a single non-covalent interaction site between two Fab domains in a characteristic "bone-like" structure observed in Transmission Electron Microscopy (TEM). These dimers showed reduced potency and antigen binding affinity. Low pH stress generated more stable but also non-covalently associated dimers without chemical alterations in a typical "closed" conformation according to TEM. These dimer species were more compact and more hydrophobic as dimers induced by process stress. They showed bioactivity and antigen binding affinity similar to the native monomer. Light-induced dimers, exhibiting various different conformations, were the most stable dimers with various chemical modifications leading to a broad range in size, charge and hydrophobicity. These dimers fully lost bioactivity and antigen binding affinity. CONCLUSION: The use of highly purified MAb dimers and a panel of characterizations methods enabled to obtain a clear picture about molecular architecture and function of dimers.

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs.

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.

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 analyte's 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.

SearchXLinks. A program for the identification of disulfide bonds in proteins from mass spectra.

We present the computer program SearchXLinks that analyzes mass spectra with the aim of identifying disulfide bonds and other modifications in proteins of known amino acid sequence. Disulfide bonds can be intra- or intermolecular. To decrease the number of false positives, the analysis of in-source decay and tandem mass spectra are coupled into the program. The steps taken during a SearchXLinks run are outlined, and the computational costs are discussed. The application of the program is illustrated by the analysis of data from recent studies on bovine ribonuclease A and bovine serum albumin. The software can be used free of charge on the Internet at http://www.searchxlinks.de.

Partial reduction and two-step modification of proteins for identification of disulfide bonds.

An experimental protocol was established to combine partial reduction, cyanylation, and a second modification step for the assignment of disulfide bonds in proteins that are resistant to proteolysis under native conditions. After proteolysis, disulfide bonds were assigned via MALDI mass spectrometry with subsequent semiautomatic interpretation using the program SearchXLinks, which enumerates all possible combinations of proteolytic fragments for all observed monoisotopic masses. The putative assignment of disulfide bonds was confirmed by ISD and PSD fragmentation of the corresponding protonated molecules.

Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS.

An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) - m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2-3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/ MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.