The Different Colors of mAbs in Solution.

The color of a therapeutic monoclonal antibody solution is a critical quality attribute. Consistency of color is typically assessed at time of release and during stability studies against preset criteria for late stage clinical and commercial products. A therapeutic protein solution's color may be determined by visual inspection or by more quantitative methods as per the different geographical area compendia. The nature and intensity of the color of a therapeutic protein solution is typically determined relative to calibrated standards. This review covers the analytical methodologies used for determining the color of a protein solution and presents an overview of protein variants and impurities known to contribute to colored recombinant therapeutic protein solutions.

Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies.

A variety of Fc domain engineering approaches for abrogating the effector functions of mAbs exists. To address some of the limitations of the current Fc domain silencing approaches, we are exploring a less commonly considered option which relies on the deletion of the hinge. Removal of the hinge domain in humanized IgG1 and IgG4 mAbs obliterates their ability to bind to activating human Fc gamma receptors I and IIIA, while leaving their ability to engage their target antigen intact. Deletion of the hinge also reduces binding to the Fc neonatal receptor, although Fc engineering allows partial recovery of affinity. Engineering of the CH3 domain, stabilizes hinge deleted IgG4s and prevents Fab arm exchange. The faster clearing properties together with the pacified Fc make modality of the hinge deleted mAb an appealing solution for therapeutic and diagnostic applications.

Isolation and characterization of a monoclonal antibody containing an extra heavy-light chain Fab arm.

Isolation and characterization of monoclonal antibody (mAb) variants to understand the impact of their structure on function is a typical activity during early-stage candidate selection that contributes to derisking clinical development. In particular, efforts are devoted to characterizing oligomeric variants, owing to their potential immunogenic nature. We report here a mAb variant consisting of a canonical mAb monomer associated in a non-covalent fashion with an antigen-binding fragment (Fab) arm amputated from its Fc domain. The truncated heavy chain is encoded in the cell line genome and is the likely product of a genomic recombination during cell line generation. The addition of the Fab arm results in severe loss of potency, indicating its interaction with the Fab domain of the monomer. The presence of such a variant can easily be mitigated by an adequate purification step.

Analytical comparability study of recombinant monoclonal antibody therapeutics.

Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle.

Quantification of a recombinant antigen in an immuno-stimulatory whole yeast cell-based therapeutic vaccine.

Therapeutic vaccines represent an emerging class of immune-modulatory treatments for cancer, infections, and chronic diseases. One such vaccine was designed as an immune stimulator of the T cell response against HBV antigens to eliminate HBV infected cells and offer a therapeutic avenue to treat patients suffering from chronic hepatitis B infection. Whole deactivated Saccharomyces cerevisiae cells expressing a recombinant fusion of HBV X, S and Core antigens elicit T cell responses in mice and activate human T cells linked with viral clearance. As the therapeutic efficacy of the yeast-based vaccine relies on the production of the recombinant antigen, analytical methods designed to accurately and precisely quantitate the fusion protein in the midst of all the yeast proteins are necessary. We report the development and characterization of western blot, quantitative ELISA and mass spectrometry based orthogonal methods to support the assessment of manufacturing consistency.

Ultrafast and high-throughput N-glycan analysis for monoclonal antibodies.

Glycosylation is a critical attribute for development and manufacturing of therapeutic monoclonal antibodies (mAbs) in the pharmaceutical industry. Conventional antibody glycan analysis is usually achieved by the 2-aminobenzamide (2-AB) hydrophilic interaction liquid chromatography (HILIC) method following the release of glycans. Although this method produces satisfactory results, it has limited use for screening a large number of samples because it requires expensive reagents and takes several hours or even days for the sample preparation. A simple and rapid glycan analysis method was not available. To overcome these constraints, we developed and compared 2 ultrafast methods for antibody glycan analysis (UMAG) that involve the rapid generation and purification of glycopeptides in either organic solvent or aqueous buffer followed by label-free quantification using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Both methods quickly yield N-glycan profiles of test antibodies similar to those obtained by the 2-AB HILIC-HPLC method. In addition, the UMAG method performed in aqueous buffer has a shorter assay time of less than 15 min, and enables high throughput analysis in 96-well PCR plates with minimal sample handling. This method, the fastest, and simplest as reported thus far, has been evaluated for glycoprofiling of mAbs expressed under various cell culture conditions, as well as for the evaluation of antibody culture clones and various production batches. Importantly the method sensitively captured changes in glycoprofiles detected by traditional 2-AB HILIC-HPLC or HILIC-UPLC. The simplicity, high speed, and low cost of this method may facilitate basic research and process development for novel mAbs and biosimilar products.

Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications.

Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography.

Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.

Enlarging the repertoire of therapeutic monoclonal antibodies platforms: domesticating half molecule exchange to produce stable IgG4 and IgG1 bispecific antibodies.

Half molecule exchange is the process whereby two IgG4 molecules exchange a heavy chain-light chain unit to form a new IgG4 entity with specificity towards two different antigens. While this unique property of IgG4 molecules confers anti-inflammatory properties in nature, it is not a desirable feature for a therapeutic mAb. Engineering of the IgG4 hinge region making it resemble that of an IgG1 is sufficient to dramatically reduce half molecule exchange in vitro and in vivo. The S228P modification of the hinge confers pharmaceutical properties to IgG4 equivalent to those of standard IgG1, while retaining the inability to trigger ADCC and CDC. Application of the molecular precepts underlying half molecule exchange between IgG4 molecules to IgG1 scaffolds offers the possibility to produce bispecific antibodies in vitro.

Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications.

Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) is a powerful analytical method used for the separation of molecular variants of therapeutic proteins. The method has been employed for monitoring various post-translational modifications, including proteolytic fragments and domain misfolding in etanercept (Enbrel®); tryptophan oxidation, aspartic acid isomerization, the formation of cyclic imide, and α amidated carboxy terminus in recombinant therapeutic monoclonal antibodies; and carboxy terminal heterogeneity and serine fucosylation in Fc and Fab fragments. HIC-HPLC is also a powerful analytical technique for the analysis of antibody-drug conjugates. Most current analytical columns, methods, and applications are described, and critical method parameters and suitability for operation in regulated environment are discussed, in this review.

Effect of pegylation on self-association of IFN-α2b.

Pegylation of therapeutic proteins is an established technology used to enhance the bioavailability of an active pharmaceutical ingredient in the body of patients. While the physiochemical properties of pegylated monomeric proteins have been extensively described, there is still limited information on the characterization of pegylated oligomeric proteins. In this study, we report the characterization of a pegylated interferon alpha2b (PEGIFN-α2b) concentration-dependent oligomerization by a series of orthogonal biochemical and biophysical methods. These methods include sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, matrix-assisted laser desorption ionization, and size exclusion chromatography of bissulfosuccinimidyl suberate cross-linked PEGIFN. We report here that PEGIFN-α2b self-associates in a concentration-dependent manner into mainly monomers, dimers, and trimers. In the presence of the chemical cross-linker, PEGIFN-α2b is primarily monomeric (57%) at concentration lower than 0.3 mg/mL and contains about equal amount of monomers and dimers (47.0% and 37.7%, respectively), about 15% of trimers, and up to 4% of higher molecular weight species at 0.7 mg/mL and above.

Developability studies before initiation of process development: improving manufacturability of monoclonal antibodies.

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2-8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.

Screening of reducing agents for the PEGylation of recombinant human IL-10.

PEGylation is a technology commonly used to enhance the bioavailability of therapeutic proteins in patients. Reductive alkylation of a protein amino terminal alpha amine in the presence of a polyethylene glycol (PEG) chain derivatized with propionaldehyde and a reducing agent, typically sodium cyanoborohydride, is one of the technologies available to achieve quantitative and site specific PEGylation. While cyanoborohydride has proven to be a robust and efficient reagent for this type of reaction, it generates aqueous cyanide as a reaction by-product (and its corollary, the very volatile hydrogen cyanide). We report here the screening of reducing agents such as dimethylamine borane, trimethylamine borane, triethylamine borane, tert-butylamine borane, morpholine borane, pyridine borane, 2-picoline borane, and 5-ethyl-2-methyl-pyridine borane as alternatives to cyanoborohydride for the PEGylation of recombinant human IL-10. The results of our study show that pyridine borane and 2-picoline borane promote rhIL-10 PEGylation at levels comparable to those observed with cyanoborohydride.

Characterization of antibody variants during process development: the tale of incomplete processing of N-terminal secretion peptide.

Monoclonal antibodies (mAbs) have emerged as one of the most important classes of biotherapeutics, although development of these molecules is long and arduous. A production cell line must be established, and growth conditions for the cells and purification processes for the product must be optimized. Integration of the appropriate analytical strategies in these activities is the cornerstone of Quality by Design and in-process control approaches are encouraged by the Food and Drug Administration. We report here the development of a reversed phase-high performance liquid chromatography (RP-HPLC) method to follow the presence of a mAb product-related variant observed during the purification process development. The variant eluted as a later peak on RP-HPLC, compared with the mAb control (3.25 min and 2.85 min, respectively). We isolated this hydrophobic variant and further analyzed it by mass spectrometry. We identified the variant as a mAb with an incompletely processed leader sequence attached to the N-terminus of one of the two heavy chains.

Efficient, chemoenzymatic process for manufacture of the Boceprevir bicyclic [3.1.0]proline intermediate based on amine oxidase-catalyzed desymmetrization.

The key structural feature in Boceprevir, Merck's new drug treatment for hepatitis C, is the bicyclic [3.1.0]proline moiety "P2". During the discovery and development stages, the P2 fragment was produced by a classical resolution approach. As the drug candidate advanced through clinical trials and approached regulatory approval and commercialization, Codexis and Schering-Plough (now Merck) jointly developed a chemoenzymatic asymmetric synthesis of P2 where the net reaction was an oxidative Strecker reaction. The key part of this reaction sequence is an enzymatic oxidative desymmetrization of the prochiral amine substrate.

A bacterial ortholog of class II lysyl-tRNA synthetase activates lysine.

Aminoacyl-tRNA synthetases produce aminoacyl-tRNAs, essential substrates for accurate protein synthesis. Beyond their central role in translation some of these enzymes or their orthologs are recruited for alternative functions, not always related to their primary cellular role. We investigate here the enzymatic properties of GenX (also called PoxA and YjeA), an ortholog of bacterial class II lysyl-tRNA synthetase. GenX is present in most Gram-negative bacteria and is homologous to the catalytic core of lysyl-tRNA synthetase, but it lacks the amino terminal anticodon binding domain of the latter enzyme. We show that, in agreement with its well-conserved lysine binding site, GenX can activate in vitro l-lysine and lysine analogs, but does not acylate tRNA(Lys) or other cellular RNAs.

Translation termination in pyrrolysine-utilizing archaea.

Although some data link archaeal and eukaryotic translation, the overall mechanism of protein synthesis in archaea remains largely obscure. Both archaeal (aRF1) and eukaryotic (eRF1) single release factors recognize all three stop codons. The archaeal genus Methanosarcinaceae contains two aRF1 homologs, and also uses the UAG stop to encode the 22nd amino acid, pyrrolysine. Here we provide an analysis of the last stage of archaeal translation in pyrrolysine-utilizing species. We demonstrated that only one of two Methanosarcina barkeri aRF1 homologs possesses activity and recognizes all three stop codons. The second aRF1 homolog may have another unknown function. The mechanism of pyrrolysine incorporation in the Methanosarcinaceae is discussed.

Misacylation of pyrrolysine tRNA in vitro and in vivo.

Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine Pyl tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo.

Adding pyrrolysine to the Escherichia coli genetic code.

Pyrrolysyl-tRNA synthetase and its cognate suppressor tRNA(Pyl) mediate pyrrolysine (Pyl) insertion at in frame UAG codons. The presence of an RNA hairpin structure named Pyl insertion structure (PYLIS) downstream of the suppression site has been shown to stimulate the insertion of Pyl in archaea. We study here the impact of the presence of PYLIS on the level of Pyl and the Pyl analog N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc) incorporation using a quantitative lacZ-luc tandem reporter system in an Escherichia coli context. We show that PYLIS has no effect on the level of neither Pyl nor Cyc incorporation. Exogenously supplying our reporter system with d-ornithine significantly increases suppression efficiency, indicating that d-ornithine is a direct precursor to Pyl.

The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity.

Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNA(Pyl), which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNA(Pyl). Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNA(Pyl) with higher affinity (K(D)=0.1-1.0 microM) than D. hafniense PylRS (K(D)=5.3-6.9 microM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNA(Pyl) species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.