ABSTRACT
Therapeutic antibodies (IgG-type) contain several post-translational modifications (PTMs) whereby introducing a large heterogeneity, both structural and functional, into this class of therapeutics. Of these modifications, glycosylation in the fragment crystallizable (Fc) region is the most heterogeneous PTM, which can affect the stability of the molecule and interactions with Fc-receptors in vivo. Hence, the glycoform distribution can affect the mode of action and have implications for bioactivity, safety and efficacy of the drug. Main topics of the manuscript include: What factors influence the (Fc) glycan pattern in therapeutic antibodies and how can these glycans be characterized? How does structure of the Fc-glycan relate to function and what methods are available to characterize those functions? Although heterogeneous in their scope, the different sections are intended to combine current knowledge on structure-function correlations of IgG glycan structures with regard to Fc (effector) functions, as well as basic aspects and methodologies for their assessment.
Subject(s)
Antibodies, Monoclonal/chemistry , Protein Processing, Post-Translational , Animals , Antibodies, Monoclonal/therapeutic use , Glycosylation , Humans , Structure-Activity RelationshipABSTRACT
Oxidation of monoclonal therapeutic antibodies (mAbs) can affect binding to Fc-receptors and potentially influence pharmacokinetics or effector functions like e.g. antibody dependent cellular phagocytosis (ADCP). Recently, it has been demonstrated that binding to FcγRIIa (H131) is affected by methionine oxidation of the Fc-portion but it is currently unknown which methionine is responsible for decreased binding. We separated an oxidized IgG1 monoclonal antibody based on the oxidation state of methionine 252 and analyzed fractionated material in receptor binding experiments as well as in functional (cell-based) assays. Although the unfractionated mixture demonstrated weaker interaction/activation of the receptor, differently oxidized isolated subspecies can lead both to stronger as well as weaker binding and activation of the histidine variant of FcγRIIa.
Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin G/immunology , Methionine/immunology , Receptors, IgG/immunology , Antibodies, Monoclonal/metabolism , Antibody Affinity/immunology , Chromatography, Liquid/methods , Histidine/genetics , Histidine/immunology , Histidine/metabolism , Humans , Immunoglobulin G/metabolism , Mass Spectrometry/methods , Methionine/metabolism , Oxidation-Reduction , Protein Binding , Receptors, IgG/genetics , Receptors, IgG/metabolism , Surface Plasmon ResonanceABSTRACT
AIM: The neonatal Fc-receptor (FcRn) mediates long serum half-life of therapeutic IgG-type antibodies. This interaction represents a critical quality attribute in terms of pharmacokinetics and should be covered by respective quality control strategies. Antibodies are taken up by cells unspecifically and can bind to FcRn in early endosomes preventing lysosomal degradation and allowing release back into circulation. Reflecting this complex cycle in an in vitro assay strategy represents a challenging task. METHODOLOGY: We report the qualification of an FcRn affinity chromatographic method and, for the first time, establish a noncriticality window. We analyzed different IgG-type antibodies, subtypes, glycoforms as well as mutants. CONCLUSION: The FcRn affinity chromatographic method allows the assessment of mAb samples with respect to their pH-dependent FcRn interaction. Furthermore, the method's capabilities and current limitations are discussed.
Subject(s)
Chromatography, Affinity/methods , Histocompatibility Antigens Class I/metabolism , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Receptors, Fc/metabolism , Chromatography, Affinity/standards , Humans , Immunoglobulin G/blood , Mutation , Reference StandardsABSTRACT
Protein-based pharmaceuticals represent the fastest growing group of drugs in development in the pharmaceutical industry. One of the major challenges in the discovery, development, and distribution of biopharmaceuticals is the assessment of changes in their higher-order structure due to chemical modification. Here, we investigated the interactions of three different biochemical probes (Fab s) generated to detect conformational changes in a therapeutic IgG1 antibody (mAbX) by local hydrogen-deuterium exchange mass spectrometry (HDX-MS). We show that two of the probes target the Fc part of the antibody, whereas the third probe binds to the hinge region. Through HDX-ETD, we could distinguish specific binding patterns of the Fc -binding probes on mAbX at the amino-acid level. Preliminary surface plasmon resonance (SPR) experiments showed that these domain-selective Fab probes are sensitive to conformational changes in distinct regions of a full-length therapeutic antibody upon oxidation.
Subject(s)
Antibodies, Monoclonal/chemistry , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G , Molecular Probes , Antibodies, Monoclonal/therapeutic use , Deuterium Exchange Measurement , Drug Design , Humans , Mass Spectrometry/methods , Protein ConformationABSTRACT
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 identification of critical quality attributes (CQAs) as an important first step for QbD development of biopharmaceuticals. A systematic scientific based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. To illustrate the application of the approach and tools, a few examples from monoclonal antibodies are shown. The identification of CQAs is a continuous process and will further drive the structure and function characterization of therapeutic proteins.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Quality Control , Animals , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
PURPOSE: Protein aggregates have been discussed as a potential risk factor related to immunogenicity. Here we developed a novel human IgG transgenic (tg) mouse system expressing a mini-repertoire of human IgG1 antibodies (Abs) for the assessment of immunogenic properties of human mAb preparations. METHODS: Transgenic mice were generated using germline versions of the human Ig heavy chain γ1 (IgH-γ1), and the human Ig light chain (IgL) κ and λ genes. Only the soluble form of human IgH-γ1 was used to avoid expression of the membrane Ig-H chain and concomitant allelic exclusion of endogenous murine Ig genes. IgG1 aggregates were generated by different stress conditions such as process-related, low pH and exposure to artificial light. RESULTS: The expression of human Ig proteins induced immunological tolerance to a broad range of human IgG1 molecules in the tg mice. Immunization with IgG1 aggregates demonstrated that soluble oligomers induced by significant light-exposure and carrying neo-epitopes induced a strong immune response in tg mice. In contrast, Ab aggregates alone and monomers with neo-epitopes were not immunogenic. CONCLUSION: This mouse model is able to recognize immunogenic modifications of human IgG1. While the degree of stress-induced aggregation varies for different mAbs, our findings using a particular mAb (mAb1) demonstrate that non-covalently modified aggregates do not break tolerance, contrary to widely held opinion. The immunogenic potential of soluble aggregates of human IgG strongly depends on the presence of neo-epitopes resulting from harsh stress conditions, i.e. extensive exposure to artificial light.
Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin G/immunology , Mice, Transgenic/immunology , Protein Aggregates/immunology , Animals , Antibodies, Monoclonal/genetics , Antibody Formation , Base Sequence , Flow Cytometry , Humans , Immune Tolerance , Immunoglobulin G/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Mice, Transgenic/genetics , Molecular Sequence Data , Protein Aggregates/genetics , Stress, Psychological/immunology , TransgenesABSTRACT
Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.
Subject(s)
Complementarity Determining Regions/metabolism , Glycation End Products, Advanced/metabolism , Immunoglobulin G/metabolism , Peptide Mapping/methods , Recombinant Proteins/metabolism , Chromatography, Ion Exchange , Chromatography, Liquid , Hot Temperature , Humans , Hydrogen-Ion Concentration , Mass Spectrometry , Oxidative Stress , Protein Processing, Post-Translational , Proteolysis , Surface Plasmon ResonanceABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/chemistry , Immunoglobulin G/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Chromatography, Gel , Human Umbilical Vein Endothelial Cells , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Protein Conformation , Protein Multimerization , Receptors, IgG/immunology , Ultraviolet RaysABSTRACT
Covalent binding of acyl glucuronides to proteins is considered an initiating event for the organ toxicity of drugs containing a carboxylic acid group. An acyl glucuronide (AcMPAG) of the immunosuppressant mycophenolic acid was described and shown to form covalent adducts with plasma albumin in vivo. The aim of the present investigation was to identify AcMPAG target proteins in the liver and colon of rats treated with mycophenolate mofetil, which may contribute to a better understanding of the mechanisms responsible for the development of side effects during therapy with this drug. Mycophenolate mofetil was administered per os in to Wistar rats (40 mg/kg/day) over 21 days. Proteins in liver and colon homogenates were separated by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. AcMPAG labeled protein spots were detected by Western blotting. After in-gel tryptic digestion of the protein spots from parallel gels (n = 2), peptides were characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. Data base searching identified AcMPAG target proteins. Tryptic peptides with sufficient signal intensities were subjected to post-source decay analysis. Three proteins in the liver (ATPase/ATP synthase (alpha and beta subunits), protein disulfide isomerase A3 and selenium binding protein) and one protein in the colon (selenium binding protein) were identified as targets for AcMPAG. ATPase/ATP synthase and protein disulfide isomerase are essential proteins involved in the control of the energy and redox state of the cells, whereas the physiological role of selenium binding protein is not fully understood. This study shows for the first time the formation of adducts between tissue proteins and AcMPAG. Whether this chemical modification is associated with compromised protein function and drug toxicity remains to be investigated.
Subject(s)
Colon/chemistry , Enzyme Inhibitors/chemistry , Glucuronides/chemistry , Liver/chemistry , Mycophenolic Acid/chemistry , Proteins/chemistry , Amino Acid Sequence , Animals , Colon/drug effects , Enzyme Inhibitors/metabolism , Female , Glucuronides/metabolism , Immunosuppressive Agents/pharmacology , Liver/drug effects , Molecular Sequence Data , Mycophenolic Acid/analogs & derivatives , Mycophenolic Acid/metabolism , Mycophenolic Acid/pharmacology , Proteins/genetics , Rats , Rats, Wistar , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
OBJECTIVE: Antibodies recognizing the ubiquitous cytosolic enzyme glucose-6-phosphate isomerase (GPI) cause arthritis in the K/BxN mouse model. Studies have shown that these antibodies are not specific for rheumatoid arthritis (RA) in humans. We evaluated GPI as a target of autoantibodies in juvenile idiopathic arthritis (JIA). METHODS: We studied 324 serum and 48 synovial fluid (SF) samples from 103 patients with JIA, 36 with RA, and 8 with arthralgia and 11 controls. Anti-GPI antibodies were assessed by densitometrically evaluating immunoblots and ELISA using native and recombinant GPI. We determined the GPI activity of the soluble antigen in serum and SF. RESULTS: Although several samples contained anti-GPI-IgG antibodies, this was not specific for JIA or its subgroups, or for RA. Other proteins in the GPI preparation were also frequently recognized by antibodies. Additionally, we observed increased GPI activity in patients with the systemic manifestation of JIA, but not in other patients. Neither anti-GPI concentrations nor GPI activity were associated with disease activity. CONCLUSION: In addition to the findings in RA, our results indicate that GPI is not a general target of autoantibodies in JIA.
Subject(s)
Arthritis, Juvenile/immunology , Autoantibodies/immunology , Glucose-6-Phosphate Isomerase/immunology , Adolescent , Arthralgia/immunology , Arthritis, Juvenile/blood , Arthritis, Juvenile/physiopathology , Arthritis, Rheumatoid/immunology , Autoantibodies/blood , Case-Control Studies , Child , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoblotting , Immunoglobulin G/metabolism , Male , Osmolar Concentration , Proteins/immunology , Recombinant Proteins/immunology , Severity of Illness Index , Synovial Fluid/immunologyABSTRACT
The molecular analysis of serum is an important field for the definition of potential diagnostic markers or disease-related protein alterations. Novel proteomic technologies such as the mass spectrometric-based surface-enhanced laser desorption/ionization (SELDI) ProteinChip technique facilitate a rapid and reproducible analysis of such protein mixtures and affords the researcher a new dimension in the search for biomarkers of disease. Here, we have applied this technology to the study of a cohort of serum samples from well-characterized renal cell carcinoma patients for the identification of such proteins by comparison to healthy controls. We detected and characterized haptoglobin 1 alpha and serum amyloid alpha-1 (SAA-1) as disease related, in addition to an as-yet-unidentified marker of 10.84 kDa. Of particular note is the detection of multiple variants of SAA-1 in multiplex that have not been described in the sera of cancer patients. SAA-1 is detected as full-length protein, des-Arginine and des-Arginine/des-Serine variants at the N terminus by SELDI. In addition, we could also detect a low-abundant variant minus the first five N-terminal amino acids. Such variants may impact the function of the protein. We conclude the technique to be a reproducible, fast and simple mode for the discovery and analysis of marker proteins of disease in serum.
Subject(s)
Apolipoproteins/blood , Blood Proteins/analysis , Carcinoma, Renal Cell/blood , Kidney Neoplasms/blood , Adult , Aged , Aged, 80 and over , Enzyme-Linked Immunosorbent Assay , Female , Haptoglobins/analysis , Humans , Male , Middle Aged , Protein Array Analysis , Serum Amyloid A Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Human leukocyte antigen (HLA)-bound peptides are central for recognition of infected/transformed cells by T cells, and have formed the basis for many immunotherapy strategies. Epitopes from a given protein sequence (e.g. from viral proteins or oncoproteins) can be predicted by algorithms, as individual HLA receptors bind peptides through defined binding motifs. Peptides with the highest predicted binding score are then normally tested for their binding ability in binding assays. However, with the assays already established, only one peptide can be tested for binding per assay. This is certainly not a reflection of the in vivo situation, where several peptides generated via the major histocompatability complex (MHC)-class I processing pathway compete for HLA-receptor binding. Here, we describe the development of a method that can mimic the competition between multiple peptides for binding to a single HLA receptor molecule. We used silica nanoparticles with immobilised HLA-A2 complexes to screen HLA-A2 binder-peptides out of a known peptide mixture. The washed beads were analysed for selectively bound peptides by matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. The advantage of the system is that the bound peptides can be unambiguously identified without any prior modification (e.g. radioactive or fluorescence labelling), even from complex peptide mixtures.
Subject(s)
HLA-A2 Antigen/metabolism , Nanotechnology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Biotinylation , Humans , Hydrogen-Ion ConcentrationABSTRACT
The beta-chains of HLA-DR molecules associated with susceptibility to rheumatoid arthritis (RA) share a common amino acid sequence in their third hypervariable region at position 70-74. This shared epitope could either contribute to preferential binding of a given disease-associated peptide, be involved in disease-induction by molecular mimicry or, by binding to heat shock proteins, influence antigen presentation. It is known that the Escherichia coli M(r)70,000 heat shock protein DnaK can bind peptides from the shared epitope. Using a highly sensitive method, we show that peptides covering the third hypervariable region of associated, but also most of the non-associated HLA-DR alleles, bind to DnaK. Similar binding specificities could be found for the constitutively expressed mammalian M(r)70,000 heat shock protein Hsc73 and the inducible mammalian Hsp72. However, peptides containing the amino acid sequence DERAA, found in HLA-DR alleles and strongly associated with protection from RA, did not bind any HSP70. Thus, our results suggest a possible association of non-binding of HSP70 to HLA-DR molecules or its 70-74 fragments and protection from RA.
Subject(s)
Escherichia coli Proteins , HLA-DR Antigens/chemistry , HLA-DR Antigens/metabolism , HSP70 Heat-Shock Proteins/metabolism , Alleles , Amino Acid Sequence , Amino Acids, Acidic/chemistry , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/prevention & control , Binding Sites , HLA-DR Antigens/genetics , HLA-DRB1 Chains , HSP70 Heat-Shock Proteins/chemistry , Humans , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Protein Structure, TertiaryABSTRACT
The alpha-defensins human neutrophil peptides (HNPs)-1, -2, and -3 have been described as cytotoxic peptides with restricted expression in neutrophils and in some lymphocytes. In this study we report that HNPs-1, -2, and -3 are also expressed in renal cell carcinomas (RCCs). Several RCC lines were found to express mRNA as well as the specific peptides of HNP-1, -2, and -3 demonstrated by reverse transcriptase-polymerase chain reaction, mass spectrometric, and flow cytometric analyses. At physiological concentrations HNPs-1, -2, and -3 stimulated cell proliferation of selected RCC lines in vitro but at high concentrations were cytotoxic for all RCC lines tested. As in RCC lines, alpha-defensins were also detected in vivo in malignant epithelial cells of 31 RCC tissues in addition to their expected presence in neutrophils. In most RCC cases randomly, patchy immunostaining of alpha-defensins on epithelial cells surrounding neutrophils was seen, but in six tumors of higher grade malignancy all tumor cells were diffusely stained. Cellular necrosis observed in RCC tissues in association with extensive patches of HNP-1, -2, and -3, seemed to be related to high concentrations of alpha-defensins. The in vitro and in vivo findings suggest that alpha-defensins are frequent peptide constituents of malignant epithelial cells in RCC with a possible direct influence on tumor proliferation.