Enabling development, manufacturing, and regulatory approval of biotherapeutics through advances in mass spectrometry.

Rapid technological advances have significantly improved the capability, versatility, and robustness of mass spectrometers which has led to them playing a central role in the development, characterization, and regulatory filings of biopharmaceuticals. Their application spans the entire continuum of drug development, starting with discovery research through product development, characterization, and marketing authorization and continues well into product life cycle management. The scope of application extends beyond traditional protein characterization and includes elements like clone selection, cell culture physiology and bioprocess optimization, investigation support, and process analytical technology. More recently, advances in the MS-based multi-attribute method are enabling the introduction of MS in a cGMP environment for routine release and stability testing. While most applications of MS to date have been for monoclonal antibodies, the successes and learnings should translate to the characterization of next-gen biotherapeutics where modalities like multispecifics could be more prevalent. In this review, we describe the most significant advances in MS and correlate them to the broad spectrum of applications to biotherapeutic development. We anticipate rapid technological improvements to continue that will further accelerate widespread deployment of MS, thereby elevating our overall understanding of product quality and enabling attribute-focused product development.

Prediction of Precision for Purity Methods.

Methods that determine the relative purity of biopharmaceuticals represent the most widely used form of analysis for the pharmaceutical industry. The ability to rapidly assess method capability or the uncertainty of measurements under actual use conditions continues to present significant challenges. We have refined and applied the model of Uncertainty Based on Current Information to predict the precision of the purity measurements and compared the predicted precision to the measured variability for several different types of purity methods. The measured method variability was derived from the analysis of data sets ranging from hundreds to thousands of measurements for each different method type. The predicted precision was found to be in excellent agreement with the statistically obtained values with R2 = 0.94. This demonstration of concurrence between the predicted and actual precision provides an opportunity for streamlining laborious conventional (statically derived) assessment of method precision and leveraging the Uncertainty Based on Current Information model utilizing much smaller data sets or even a single experiment.

Application of a Quantitative LC-MS Multiattribute Method for Monitoring Site-Specific Glycan Heterogeneity on a Monoclonal Antibody Containing Two N-Linked Glycosylation Sites.

A significant challenge of traditional glycan mapping techniques is that they do not provide site-specific glycosylation information. Therefore, for proteins containing multiple glycosylation sites, the individual glycan species present at a particular site cannot be differentiated from those species present at the other glycosylation sites on the molecule. Quantification of glycoform has previously been demonstrated using a multiattribute method (MAM), which can quantify multiple post-translational modifications including deamidation, oxidation, glycosylation variants, and fragmentation ( Rogers, R. S.; Nightlinger, N. S.; Livingston, B.; Campbell, P.; Bailey, R.; Balland, A. MAbs 2015 , 7 , 881 - 890 ; ref 1). In this paper we describe the application of an MAM based method for site specific quantification of N-linked glycan heterogeneity present on an IgG1 mAb molecule containing two distinct N-linked glycosylation sites: one present on the heavy chain (HC) variable region (Fab) and the other present on the conserved HC constant region (Fc). MAM is a peptide mapping method utilizing mass spectrometry to detect and quantify specific peptides of interest. The ionization properties of the glycopeptides with different classes of glycan structural variants, including high mannose, sialylated, and terminal galactosylated species were studied in detail. Our results demonstrate that MAM quantification of individual glycan species from both the Fab and Fc N-Linked glycosylation sites is consistent with quantification using the traditional hydrophilic interaction liquid chromatography (HILIC) analysis of enzymatically released and fluorescently labeled glycans. Furthermore, no significant impact from the glycoform on the ionization properties of the glycopeptide is observed. Our work demonstrates that the MAM method is a suitable approach for providing quantitative, site-specific glycan information for profiling of N-linked glycans on immunoglobulins.

Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

Monoclonal antibody disulfide reduction during manufacturing: Untangling process effects from product effects.

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.

Assessment of stable isotope incorporation into recombinant proteins.

Stable isotope labeling combined with mass spectrometry has been widely used in a diverse set of applications in the biochemistry and biomedical fields. When stable isotope-labeled proteins are produced via metabolic labeling of cell culture, a comprehensive assessment of the labeling pattern is imperative. In this study, we present a set of mass spectrometry-based bioanalytical tools developed for quantitatively tracing the levels of the stable isotopes incorporated into the recombinant proteins (monoclonal antibodies and Fc fusion proteins expressed in different host systems) that include total mass analysis, peptide mapping analysis, and amino acid analysis. We show that these three mass spectrometry-based analytical methods have distinctive advantages and limitations and that they are mutually complementary in evaluating the quality of stable isotope-labeled proteins. In addition, we show that the analytical techniques developed here are powerful tools to provide valuable insights into studying cell metabolism and performing flux analysis during cell culture.

Analysis and characterization of aggregation of a therapeutic Fc-fusion protein.

Protein aggregation was observed in a purification intermediate of a therapeutic Fc-fusion protein stored at -30 °C, even though the protein was stable at 4 and -80 °C. The protein was expressed in Escherichia coli as an inclusion body, refolded, and purified using chromatography columns. To study the nature of this aggregation, a series of experiments were conducted to investigate factors that contributed to the protein instability during freezing. We found that the presence of free thiols in the protein is the intrinsic cause. The free thiol cross-linking sites were determined to be at the peptide moiety of the Fc-fusion protein using LC-MS. Partially frozen accompanied by the elevated pH and increased salt and protein concentrations were identified as extrinsic factors that facilitated the aggregation. These results provided important insights into purification process improvement and solution storage of this Fc-fusion protein.

Uncertainty estimates of purity measurements based on current information: toward a "live validation" of purity methods.

PURPOSE: To predict precision and other performance characteristics of chromatographic purity methods, which represent the most widely used form of analysis in the biopharmaceutical industry. METHODS: We have conducted a comprehensive survey of purity methods, and show that all performance characteristics fall within narrow measurement ranges. This observation was used to develop a model called Uncertainty Based on Current Information (UBCI), which expresses these performance characteristics as a function of the signal and noise levels, hardware specifications, and software settings. RESULTS: We applied the UCBI model to assess the uncertainty of purity measurements, and compared the results to those from conventional qualification. We demonstrated that the UBCI model is suitable to dynamically assess method performance characteristics, based on information extracted from individual chromatograms. CONCLUSIONS: The model provides an opportunity for streamlining qualification and validation studies by implementing a "live validation" of test results utilizing UBCI as a concurrent assessment of measurement uncertainty. Therefore, UBCI can potentially mitigate the challenges associated with laborious conventional method validation and facilitates the introduction of more advanced analytical technologies during the method lifecycle.

Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. II. Performance.

In this report, we examine the performance of a mass spectrometry (MS)-based method for quantification of protein posttranslational modifications (PTMs) using stable isotope labeled internal standards. Uniform labeling of proteins and highly similar behavior of the labeled vs nonlabeled analyte pairs during chromatographic separation and electrospray ionization (ESI) provide the means to directly quantify a wide range of PTMs. In the companion report (Jiang et al., Anal. Biochem., 421 (2012) 506-516.), we provided principles and example applications of the method. Here we show satisfactory accuracy and precision for quantifying protein modifications by using the SILIS method when the analyses were performed on different types of mass spectrometers, such as ion-trap, time-of-flight (TOF), and quadrupole instruments. Additionally, the stable isotope labeled internal standard (SILIS) method demonstrated an extended linear range of quantification expressed in accurate quantification up to at least a 4 log concentration range on three different types of mass spectrometers. We also demonstrate that lengthy chromatographic separation is no longer required to obtain quality results, offering an opportunity to significantly shorten the method run time. The results indicate the potential of this methodology for rapid and large-scale assessment of multiple quality attributes of a therapeutic protein in a single analysis.

Quantification of protein posttranslational modifications using stable isotope and mass spectrometry I: principles and applications.

With the increased attention to quality by design (QbD) for biopharmaceutical products, there is a demand for accurate and precise quantification methods to monitor critical quality attributes (CQAs). To address this need we have developed a mass spectrometry (MS) based method to quantify a wide range of posttranslational modifications (PTMs) in recombinant proteins using stable isotope-labeled internal standard (SILIS). The SILIS was produced through metabolic labeling where ¹5N was uniformly introduced at every nitrogen atom in the studied proteins. To enhance the accuracy of the method, the levels of PTMs in SILIS were quantified using orthogonal analytical techniques. Digestion of an unknown sample mixed with SILIS generates a labeled and a nonlabeled version of each peptide. The nonlabeled and labeled counterparts coelute during RP-HPLC separation but exhibit a sufficient mass difference to be distinguished by MS detection. With the application of SILIS, numerous PTMs can be quantified in a single analysis based on the measured MS signal ratios of ¹5N-labeled versus the nonlabeled pairs. Several examples using microbial and mammalian-expressed recombinant proteins demonstrated the principle and utility of this method. The results indicate that SILIS is a valuable methodology in addressing CQAs for the QbD paradigm.

Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process.

Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used during the CE-SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE-SDS versus SDS-PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.

Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography.

Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn.

Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.

Comparison of different approaches for evaluation of the detection and quantitation limits of a purity method: a case study using a capillary isoelectrofocusing method for a monoclonal antibody.

Several different techniques suggested by the International Conference on Harmonization (ICH) Q2R1 guideline were used to assess the signal and concentration at the limit of detection (LOD) and limit of quantitation (LOQ) for a purity method. These approaches were exemplified with a capillary isoelectrofocusing (cIEF) method, which has been developed to quantify the distribution of the charge isoforms of a monoclonal antibody. The charge isoforms are the result of incomplete posttranslational processing of C-terminal lysine residues of the heavy chain by carboxypeptidase. Results showed no significant discrepancy between LOD/LOQ obtained by the different techniques. Validation experiments corroborated the calculated LOQ. The results indicate that any single technique can provide meaningful values for the LOD and LOQ. Finally, important points to consider when applying these techniques to purity methods are discussed.