Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations.

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (CNaCl) and monoclonal antibody (mAb) concentration (CmAb) were assessed using the diffusion interaction parameter kD and second virial coefficient B22 measured from solutions with low to moderate CmAb. The effective structure factor S(q)eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with CNaCl, but also with increasing CmAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including kD and S(q)eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low CmAb.

Control of Antibody Impurities Induced by Riboflavin in Culture Media During Production.

During the manufacturing of protein biologics, product variability during cell culture production and harvest needs to be actively controlled and monitored to maintain acceptable product quality. To a large degree, variants that have previously been described are covalent in nature and are easily analyzed by a variety of techniques. Here, we describe a noncovalent post translational modification of recombinantly expressed antibodies, containing variable domain tryptophans, that are exposed to culture media components and ambient laboratory light. The modified species, designated as conformer, can be monitored by hydrophobic interaction chromatography and often exhibits reduced potency. We studied conformer formation and identified key elements driving its accelerated growth using an IgG2 monoclonal antibody. Conformer is a result of a noncovalent interaction of the antibody with riboflavin, an essential vitamin added to many production cell culture formulations. Chemical and physical factors that influence the impact of riboflavin are identified, and methods for process control of this product quality attribute are addressed in order to prevent loss of antibody potency and potential safety issues. Identifying therapeutic antibody drug candidates with the potential to form conformers can be performed early in development to avoid this undesirable product quality propensity.

Structural Changes and Aggregation Mechanisms of Two Different Dimers of an IgG2 Monoclonal Antibody.

Protein therapeutics, monoclonal antibodies (mAbs) in particular, are large, structurally complex molecules that are prone to numerous modes of degradation during their production and long-term storage. Physical degradation via protein aggregation is a major concern when developing protein therapeutic candidates for clinical use. A dimer is perhaps the simplest element of protein aggregation, and thus, a better understanding of protein dimers in terms of their structures, intermolecular interactions, and chemical nature will help in the development of rational strategies for reducing aggregation propensity. In this study, two different mAb dimers were generated from an IgG2 monoclonal antibody solution, i.e., a native dimer generated under long-term storage and a thermal dimer from a thermal stress condition. Both IgG2 dimers were characterized in terms of their chemical and physical properties, bioactivity, and conformational dynamics. The native IgG2 dimer was formed mainly through noncovalent association. It displayed minimal differences in biophysical properties and higher-order structure compared to the monomer yet showed compromised in vitro potency, likely because of steric hindrance. In contrast, the thermal IgG2 dimer was mainly disulfide-linked, but even so, no new non-native disulfide bonds were detected by peptide mapping. Two regions within the Fc-CH2 domain of the thermal IgG2 dimer exhibited significantly increased flexibility as measured by hydrogen-deuterium exchange mass spectrometry, and notably, these regions are connected by an intrachain disulfide bond under natively folded conditions. These findings provide a better understanding of dimer formation under long-term storage and thermal stress conditions for this IgG2 mAb, and possible aggregation mechanisms are discussed.

High-Throughput Screening and Analysis of Charge Variants of Monoclonal Antibodies in Multiple Formulations.

Among different biopharmaceutical products, monoclonal antibodies (mAbs) show a high level of complexity, including heterogeneity due to differences in size, hydrophobicity, charge, and so forth. Such heterogeneity can be related to both cell-based production and any of the stages of purification, storage, and delivery that the mAb is subjected to. Choosing the right formulation composition providing both physical and chemical stabilities can be a very challenging process, especially when done in the limited time frame required for a typical drug development cycle. Charge variants, a common type of heterogeneity for mAbs, are easy to detect by ion exchange, specifically cation exchange chromatography (CEX). We have developed and implemented a high-throughput CEX-based approach for the rapid screening and analysis of charge modifications in multiple formulation conditions. In this work, 96 different formulations of antistreptavidin IgG1 and IgG2 molecules were automatically prepared and analyzed after incubation at high temperature. Design of experiment and statistical analysis tools have been utilized to determine the major formulation factors responsible for chemical stability of antibodies. Regression models were constructed to find the optimal formulation conditions. The methodology can be applied to different stages of preformulation and formulation development of mAbs.

Effects of sucrose and benzyl alcohol on GCSF conformational dynamics revealed by hydrogen deuterium exchange mass spectrometry.

Protein stability, one of the major concerns for therapeutic protein development, can be optimized during process development by evaluating multiple formulation conditions. This can be a costly and lengthy procedure where different excipients and storage conditions are tested for their impact on protein stability. A better understanding of the effects of different formulation conditions at the molecular level will provide information on the local interactions within the protein leading to a more rational design of stable and efficacious formulations. In this study, we examined the roles of the excipients, sucrose and benzyl alcohol, on the conformational dynamics of recombinant human granulocyte colony stimulating factor using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS). Under physiological pH and temperature, sucrose globally protects the whole molecule from deuterium uptake, whereas benzyl alcohol induces increased deuterium uptake of the regions within the α-helical bundle, with even larger extent. The HDX experiments described were incorporated a set of internal peptides (Zhang et al., 2012. Anal Chem 84:4942-4949) to monitor the differences in intrinsic exchange rates in different formulations. In addition, we discussed the feasibility of implementing HDX-MS with these peptide probes in protein formulation development.

Accelerated formulation development of monoclonal antibodies (mAbs) and mAb-based modalities: review of methods and tools.

More therapeutic monoclonal antibodies and antibody-based modalities are in development today than ever before, and a faster and more accurate drug discovery process will ensure that the number of candidates coming to the biopharmaceutical pipeline will increase in the future. The process of drug product development and, specifically, formulation development is a critical bottleneck on the way from candidate selection to fully commercialized medicines. This article reviews the latest advances in methods of formulation screening, which allow not only the high-throughput selection of the most suitable formulation but also the prediction of stability properties under manufacturing and long-term storage conditions. We describe how the combination of automation technologies and high-throughput assays creates the opportunity to streamline the formulation development process starting from early preformulation screening through to commercial formulation development. The application of quality by design (QbD) concepts and modern statistical tools are also shown here to be very effective in accelerated formulation development of both typical antibodies and complex modalities derived from them.

Optimized UV detection of high-concentration antibody formulations using high-throughput SE-HPLC.

High-concentration antibody solutions (>100 mg/mL) present significant challenges for formulation and process development, including formulation attributes such as increased solution viscosity, and the propensity for self-association. An additional challenge comes from the adaptation of analytical methods designed for low-concentration formulations to the high-concentration regime. The oligomeric state is a good example: it is a quality attribute monitored during pharmaceutical development and is one that can be affected by dilution; a typical first step in the analysis of high-concentration solutions. The objective of this work was to develop a size-exclusion HPLC (SE-HPLC) method that would allow the injection of high-concentration antibody formulations without the need for dilution prior to injection and their analysis in a high-throughput manner that does not create a bottleneck for the execution of complex formulation development studies. It was found that changing the UV detection wavelength from 215 to 235 nm simplified sample preparation by allowing for an approximately fivefold increase in injection load while maintaining the signal within the linear range of detection. In addition, the chromatographic peak properties (i.e., peak symmetry, resolution, and sensitivity) were determined to be consistent when compared with analytical methods developed for formulations with lower antibody concentrations.

High-throughput screening and stability optimization of anti-streptavidin IgG1 and IgG2 formulations.

Selection of a suitable formulation that provides adequate product stability is an important aspect of the development of biopharmaceutical products. Stability of proteins includes not only resistance to chemical modifications but also conformational and colloidal stabilities. While chemical degradation of antibodies is relatively easy to detect and control, propensity for conformational changes and/or aggregation during manufacturing or long-term storage is difficult to predict. In many cases, the formulation factors that increase one type of stability may significantly decrease another type under the same or different conditions. Often compromise is necessary to minimize the adverse effects of an antibody formulation by careful optimization of multiple factors responsible for overall stability. In this study, high-throughput stress and characterization techniques were applied to 96 formulations of anti-streptavidin antibodies (an IgG1 and an IgG2) to choose optimal formulations. Stress and analytical methods applied in this study were 96-well plate based using an automated liquid handling system to prepare the different formulations and sample plates. Aggregation and clipping propensity were evaluated by temperature and mechanical stresses. Multivariate regression analysis of high-throughput data was performed to find statistically significant formulation factors that alter measured parameters such as monomer percentage or unfolding temperature. The results of the regression models were used to maximize the stabilities of antibodies under different formulations and to find the optimal formulation space for each molecule. Comparison of the IgG1 and IgG2 data indicated an overall greater stability of the IgG1 molecule under the conditions studied. The described method can easily be applied to both initial preformulation screening and late-stage formulation development of biopharmaceutical products.

Small-angle neutron scattering study of a monoclonal antibody using free-energy constraints.

Monoclonal antibodies (mAbs) contain hinge-like regions that enable structural flexibility of globular domains that have a direct effect on biological function. A subclass of mAbs, IgG2, have several interchain disulfide bonds in the hinge region that could potentially limit structural flexibility of the globular domains and affect the overall configuration space available to the mAb. We have characterized human IgG2 mAb in solution via small-angle neutron scattering (SANS) and interpreted the scattering data using atomistic models. Molecular Monte Carlo combined with molecular dynamics simulations of a model mAb indicate that a wide range of structural configurations are plausible, spanning radius of gyration values from ∼39 to ∼55 Å. Structural ensembles and representative single structure solutions were derived by comparison of theoretical SANS profiles of mAb models to experimental SANS data. Additionally, molecular mechanical and solvation free-energy calculations were carried out on the ensemble of best-fitting mAb structures. The results of this study indicate that low-resolution techniques like small-angle scattering combined with atomistic molecular simulations with free-energy analysis may be helpful to determine the types of intramolecular interactions that influence function and could lead to deleterious changes to mAb structure. This methodology will be useful to analyze small-angle scattering data of many macromolecular systems.

Methods of high throughput biophysical characterization in biopharmaceutical development.

Discovery and successful development of biopharmaceutical products depend on a thorough characterization of the molecule both before and after formulation. Characterization of a formulated biotherapeutic, typically a protein or large peptide, requires a rigorous assessment of the molecule's physical stability. Stability of a biotherapeutic includes not only chemical stability, i.e., degradation of the molecule to form undesired modifications, but also structural stability, including the formation of aggregates. In this review, high throughput biophysical characterization techniques are described according to their specific applications during biopharmaceutical discovery, development and manufacturing. The methods presented here are classified according to these attributes, and include spectroscopic assays based on absorbance, polarization, intrinsic and extrinsic fluorescence, surface plasmon resonance instrumentation, calorimetric methods, dynamic and static light scattering techniques, several visible particle counting and sizing methods, new viscosity assay, based on light scattering and mass spectrometry. Several techniques presented here are already implemented in industry; but, many high throughput biophysical methods are still in the initial stages of implementation or even in the prototype stage. Each technique in this report is judged by the specific application of the method through the biopharmaceutical development process.

Characterization of site-specific glycation during process development of a human therapeutic monoclonal antibody.

A therapeutic recombinant monoclonal antibody (mAb1) was found to be highly susceptible to glycation during production. Up to 42% glycation was observed in mAb1, which was significantly greater than the glycation observed in 17 other monoclonal antibodies (mAbs). The majority of the glycation was localized to lysine 98 of a unique sequence in the heavy chain complementarity determining region 3. Upon incubation with 5% glucose at 37 °C for 5 days, the level of glycation rose to 80% of the total protein where the majority of the additional glycation was on the lysine 98 residue. These data suggested that the lysine 98 residue was highly susceptible to glycation. However, three other mAbs with a lysine residue in the same position did not show high rates of glycation in the forced glycation assay, suggesting that primary and perhaps secondary structural constraints could contribute to the rate of glycation at that lysine. Interestingly, a portion of the glycation in mAb1 was found to be reversible and upon incubation in phosphate buffer (pH 7) at 37 °C for 5 days, the glycation dropped from starting levels of 42% to 20%. Variation was observed in the total glycation levels between different lots of mAb1. The variability in glycation introduced charge heterogeneity in the form of an acidic peak on cation exchange chromatography and lead to product inconsistency. Mutation of lysine 98 to arginine reduced the starting level of glycation without any impact on potency.

Middle-down fragmentation for the identification and quantitation of site-specific methionine oxidation in an IgG1 molecule.

A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middle-down N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.

Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics.

Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled (13)C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified-free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from (13)C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO(2) through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.

Glutamine-linked and non-consensus asparagine-linked oligosaccharides present in human recombinant antibodies define novel protein glycosylation motifs.

We report the presence of oligosaccharide structures on a glutamine residue present in the V(L) domain sequence of a recombinant human IgG2 molecule. Residue Gln-106, present in the QGT sequence following the rule of an asparagine-linked consensus motif, was modified with biantennary fucosylated oligosaccharide structures. In addition to the glycosylated glutamine, analysis of a lectin-enriched antibody population showed that 4 asparagine residues: heavy chain Asn-162, Asn-360, and light chain Asn-164, both of which are present in the IgG1 and IgG2 constant domain sequences, and Asn-35, which was present in CDR(L)1, were also modified with oligosaccharide structures at low levels. The primary sequences around these modified residues do not adhere to the N-linked consensus sequon, NX(S/T). Modeling of these residues from known antibody crystal structures and sequence homology comparison indicates that non-consensus glycosylation occurs on Asn residues in the context of a reverse consensus motif (S/T)XN located on highly flexile turns within 3 residues of a conformational change. Taken together our results indicate that protein glycosylation is governed by more diversified requirements than previously appreciated.

LC/MS analysis of complex multiglycosylated human alpha(1)-acid glycoprotein as a model for developing identification and quantitation methods for intact glycopeptide analysis.

The site-specific characterization of the complex glycans in multiglycosylated proteins requires developing methods where the carbohydrates remain covalently bound to the protein. The complexity in the carbohydrate composition of alpha(1)-acid glycoprotein (AAG) makes it an ideal model protein for such development. AAG has five N-asparaginyl-linked glycosylation sites, each varying in its bi-, tri-, and tetraantennary glycan content. We present an on-line liquid chromatography/mass spectrometry (LC/MS) method that uses high-low cone voltage switching for in-source fragmentation to determine the structures of the complex glycans present on each site for the two gene products of AAG. High cone voltage caused carbohydrate fragmentation, leading to the generation of signature carbohydrate ions that we used as markers to identify the glycopeptides. Low cone voltage produced minimal carbohydrate fragmentation and enabled the identification and quantification of the intact oligosaccharide structures on each glycopeptide based on its monoisotopic mass and intensity. Quantitation was accomplished by using the intensities of peaks from deconvoluted and deisotoped mass spectra or from the areas of the extracted ion chromatograms from the tryptic peptide maps. The combined results from the two methods can be used to better characterize and quantitate site heterogeneity in multiglycosylated proteins.

Asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the CH1 domain of human antibodies.

We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the C(H)1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass. HPLC-MS results indicated that cation-exchange chromatography acidic variant populations were enriched in antibody with a second glycosylation site, in addition to the well documented canonical glycosylation site located in the C(H)2 domain. Subsequent tryptic and chymotryptic peptide map data indicated that the second glycosylation site was associated with the amino acid sequence TVSWN(162)SGAL in the C(H)1 domain of the antibody. This highly atypical modification is present at levels of 0.5-2.0% on most of the recombinant antibodies that have been tested and has also been observed in IgG1 antibodies derived from human donors. Site-directed mutagenesis of the C(H)1 domain sequence in a recombinant-human IgG1 antibody resulted in an increase in non-consensus glycosylation to 3.15%, a greater than 4-fold increase over the level observed in the wild type, by changing the -1 and +1 amino acids relative to the asparagine residue at position 162. We believe that further understanding of the phenomenon of non-consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery.

An improved trypsin digestion method minimizes digestion-induced modifications on proteins.

Trypsin digestion can induce artificial modifications such as asparagine deamidation and N-terminal glutamine cyclization on proteins due to the temperature and the alkaline pH buffers used during digestion. The amount of these artificial modifications is directly proportional to the incubation time of protein samples in the reduction/alkylation buffer and, more important, in the digestion buffer where the peptides are completely solvent exposed. To minimize these artificial modifications, we focused on minimizing the trypsin digestion time by maximizing trypsin activity. Trypsin activity was optimized by the complete removal of guanidine, which is a known trypsin inhibitor, from the digestion buffer. As a result, near complete trypsin digestion was achieved on reduced and alkylated immunoglobulin gamma molecules in 30min. The protein tryptic fragments and their modification products were analyzed and quantified by reversed-phase liquid chromatography/tandem mass spectrometry using an in-line LTQ Orbitrap mass spectrometer. The reduction and alkylation reaction time was also minimized by monitoring the completeness of the reaction using a high-resolution time-of-flight mass spectrometer. Using this 30-min in-solution trypsin digestion method, little protocol-induced deamidation or N-terminal glutamine cyclization product was observed and cleaner tryptic maps were obtained due to less trypsin self-digestion and fewer nonspecific cleavages. The throughput of trypsin digestion was also improved significantly compared with conventional trypsin digestion methods.

O-fucosylation of an antibody light chain: characterization of a modification occurring on an IgG1 molecule.

We describe the characterization of an O-fucosyl modification to a serine residue on the light chain of a recombinant, human IgG1 molecule expressed in Chinese hamster ovary (CHO) cells. Cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC) were used to isolate a Fab population which was 146 Da heavier than the expected mass. Isolated Fab fragments were treated with a reducing agent to facilitate mass spectrometric analysis of the reduced light chain (LC) and fragment difficult (Fd). An antibody light chain with a net addition of 146 Da was detected by mass spectrometric analysis of the modified Fab. A light chain tryptic peptide in complementarity determining region-1 (CDR-1) was subsequently identified with a net addition of 146 Da by a peptide map. Results from a nanospray infusion of the modified peptide into a linear ion trap mass spectrometer with electron transfer dissociation (ETD) functionality indicated that the modified residue was a serine at position 30 in the light chain. Acid hydrolysis of the modified tryptic peptide followed by fluorescent labeling with 2-aminoanthranilic acid (2AA) and HPLC comparison with monosaccharide standards confirmed the presence of fucose on the light chain peptide. The presence of O-fucose on an antibody has not been previously reported. Currently, O-fucose has been described as occurring on mammalian proteins with amino acid sequence motifs associated with epidermal growth factor (EGF)-like repeats or thrombospondin type 1 repeats (TSRs). The amino acid sequence around the modified Ser in the IgG1 molecule does not conform to any known O-fucosylation sequence motif and thus is the first description of this type of modification on a nonconsensus sequence in a mammalian protein.

Degradation products analysis of an Fc fusion protein using LC/MS methods.

The following analytical methods have been used to identify and quantify degradation products in an E. coli expressed human immunoglobulin G Fc fusion protein in both liquid and lyophilized forms: two-dimensional AEX/RP/MS, limited proteolysis followed by LC/MS, and tryptic digestion followed by LC/MS/MS. After aging in a potassium phosphate pH 7.0 buffer for 3 months at 29 degrees C, peptide map analysis revealed that asparagine N78 (N297 according to Edelman sequencing) of the CH2 domain was the most rapidly deamidated site in the molecule probably due to the lack of the N-linked glycan on this asparagine, but this deamidation can be prevented under properly formulated conditions. This is the first report on the rate of deamidation on N297 of an IgG molecule without glycosylation. The active protein portion of the Fc fusion protein contains two methionine residues that are potentially susceptible to oxidation. Limited proteolysis was employed to cleave the active protein portion and measure the amount of oxidation. LC/MS analysis identified that the liquid sample aged at 29 degrees C for 3 months produced 40% oxidation, while the control sample contained only 4% oxidation on the active protein. In contrast to the aged liquid sample, the aged lyophilized sample showed no increase of deamidation or oxidation after storage at 37 degrees C for 8 months.

Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry.

An N-terminal top-down sequencing approach was developed for IgG characterization, using high-resolution HPLC separation and collisionally activated dissociation (CAD) on a single-stage LCT Premier time of flight (TOF) mass spectrometer. Fragmentation of the IgG chains on the LCT Premier was optimized by varying the ion guide voltage values. Ion guide 1 voltage had the most significant effect on the fragmentation of the IgG chains. An ion guide 1 voltage value of 100 V was found to be optimum for the N-terminal fragmentation of IgG heavy and light chains, which are approximately 50 and 25 kDa, respectively. The most prominent ion series in this CAD experiment was the terminal b-ion series which allows N-terminal sequencing. Using this technique, we were able to confirm the sequence of up to seven N-terminal residues. Applications of this method for the identification of N-terminal pyroglutamic acid formation will be discussed. The method described could be used as a high-throughput method for the rapid N-terminal sequencing of IgG chains and for the detection of chemical modifications in the terminal residues.