Influence of glycosylation pattern on the molecular properties of monoclonal antibodies

Glycosylation is an important post-translational modification during protein production in eukaryotic cells, and it is essential for protein structure, stability, half-life, and biological functions. In this study, we produced various monoclonal antibody (mAb) glycoforms from Chinese hamster ovary (CHO) cells, including the natively glycosylated antibody, the enriched G0 form, the deglycosylated form, the afucosylated form, and the high mannose form, and we compared their intrinsic properties, side-by-side, through biophysical and biochemical approaches. Spectroscopic analysis indicates no measureable secondary or tertiary structural changes after in vitro or in vivo modification of the glycosylation pattern. Thermal unfolding experiments show that the high mannose and deglycosylated forms have reduced thermal stability of the CH2 domain compared with the other tested glycoforms. We also observed that the individual domain’s thermal stability could be pH dependent. Proteolysis analysis indicates that glycosylation plays an important role in stabilizing mAbs against proteases. The stability of antibody glycoforms at the storage condition (2–8 °C) and at accelerated conditions (30 and 40 °C) was evaluated, and the results indicate that glycosylation patterns do not substantially affect the storage stability of the antibody we studied.     

Understanding the relevance of local conformational stability and dynamics to the aggregation propensity of an IgG1 and IgG2 monoclonal antibodies

Aggregation of monoclonal antibodies is often a multi‐step process involving structural alterations in monomeric proteins and subsequent formation of soluble or insoluble oligomers. The role of local conformational stability and dynamics of native and/or partially altered structures in determining the aggregation propensity of monoclonal antibodies, however, is not well understood. Here, we investigate the role of conformational stability and dynamics of regions with distinct solvent exposure in determining the aggregation propensity of an IgG1 and IgG2 monoclonal antibody. The temperatures employed span the pre‐unfolding range (10–40°C) and the onset temperatures (T_onset) for exposure of apolar residues (～50°C), alterations in secondary structures (～60°C) and initiation of visible aggregate formation (～60°C). Solvent‐exposed regions were found to precede solvent‐shielded regions in an initiation of aggregation for both proteins. Such a process was observed upon alterations in overall tertiary structure while retaining the secondary structures in both the proteins. In addition, a greater dynamic nature of solvent‐shielded regions in potential intermediates of IgG1 and the improved conformational stability increased its resistance to aggregation when compared to IgG2. These results suggest that local conformational stability and fluctuations of partially altered structures can influence the aggregation propensity of immunoglobulins.     

Production of monoclonal antibodies with a controlled N‐glycosylation pattern in seeds of Arabidopsis thaliana

Seed‐specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild‐type (wt) plants and glycosylation mutants lacking plant specific N‐glycan residues. We demonstrate that 2G12 is produced with complex N‐glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N‐glycans significant amounts of oligo‐mannosidic structures, which are typical for endoplasmic reticulum (ER)‐retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDEL‐tagged version of 2G12 exhibited an ER‐typical N‐glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N‐glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.     

Fabrication and characterization of noble crystalline silver nanoparticles from Pimenta dioica leave extract and analysis of chemical constituents for larvicidal applications

The current works report the bio-efficacy of Pimenta dioica leaf derived silver nanoparticles (Pd@AgNPs) and leaf extract obtained trough different solvents against the larvae of malaria, filarial and dengue vectors. Synthesis of silver nanoparticles (AgNPs) was done by adding 10 ml of P. dioica leaf extract into 90 ml of 1 mM silver nitrate solution, a slow colour change was observed depicting the formation of AgNPs. Further, Pd@AgNPs was confirmed through Ultraviolet–visible spectroscopy which exhibited characteristic absorption peak at 422 nm wavelength. X-ray diffraction and selected area electron diffraction analysis confirmed monodispersed and crystalline nature of Pd@AgNPs with 32 nm an average size. Scanning electron microscopy and transmission electron microscopy showed the most of Pd@AgNPs were spherical and triangular in shape and energy-dispersive X-ray spectroscopy revealed silver elemental nature of nanoparticles. Zeta potential of Pd@AgNPs is highly negative which confirmed its stable nature. Pd@AgNPs showed prominent absorption peaks at 1015, 1047, 1243, 1634, 2347, 2373, 2697 and 3840 cm^?1 which are corresponding to following compounds polysaccharides, carboxylic acids, water, alcohols, esters, ethers, amines, amides and phenol, respectively as reported by Fourier-transform infrared spectroscopy analysis. Gas chromatography–mass spectrometry and Liquid chromatography–mass spectrometry analysis revealed 39 and 70 compounds, respectively, which might be contributed for bio-reduction, capping, stabilization and larvicidal behavior of AgNPs. A comparable lethality (LC₅₀ and LC₉₀) was observed in case of Pd@AgNPs over leaf extract alone. The potential larvicidal activity of Pd@AgNPs was observed against the larvae of Aedes aegypti,(LC_50, 2.605; LC_90, 5.084 ppm) Anopheles stephensi (LC_50, 3.269; LC_90, 7.790 ppm) and Culex quinquefasciatus (LC_50, 5.373; LC_90, 14.738 ppm without affecting non-targeted organism, Mesocyclops thermocyclopoides after 72 hr of exposure. This study entails green chemistry behind synthesis of AgNPs which offers effective technique for mosquito control and other therapeutic applications.     

Effects of subclass change on the structural stability of chimeric,humanized, and human antibodies under thermal stress

To address how changes in the subclass of antibody molecules affect their thermodynamic stability, we prepared three types of four monoclonal antibody molecules (chimeric, humanized, and human) and analyzed their structural stability under thermal stress by using size‐exclusion chromatography, differential scanning calorimetry (DSC), circular dichroism (CD), and differential scanning fluoroscopy (DSF) with SYPRO Orange as a dye probe. All four molecules showed the same trend in change of structural stability; the order of the total amount of aggregates was IgG1 < IgG2 < IgG4. We thus successfully cross‐validated the effects of subclass change on the structural stability of antibodies under thermal stress by using four methods. The T_h values obtained with DSF were well correlated with the onset temperatures obtained with DSC and CD, suggesting that structural perturbation of the CH2 region could be monitored by using DSF. Our results suggested that variable domains dominated changes in structural stability and that the physicochemical properties of the constant regions of IgG were not altered, regardless of the variable regions fused.     

Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns

Quality by design (QbD) is a scheme for the development, manufacture, and approval of pharmaceutical products. The end goal of QbD is to ensure product quality by building it into the manufacturing process. The main regulatory bodies are encouraging its implementation to the manufacture of all new pharmaceuticals including biological products. Monoclonal antibodies (mAbs) are currently the leading products of the biopharmaceutical industry. It has been widely reported that glycosylation directly influences the therapeutic mechanisms by which mAbs function in vivo. In addition, glycosylation has been identified as one of the main sources of monoclonal antibody heterogeneity, and thus, a critical parameter to follow during mAb manufacture. This article reviews the research on glycosylation of mAbs over the past 2 decades under the QbD scope. The categories presented under this scope are: (a) definition of the desired clinical effects of mAbs, (b) definition of the glycosylation‐associated critical quality attributes (glycCQAs) of mAbs, (c) assessment of process parameters that pose a risk for mAb glycCQAs, and (d) methods for accurately quantifying glycCQAs of mAbs. The information available in all four areas leads us to conclude that implementation of QbD to the manufacture of mAbs with specific glycosylation patterns will be a reality in the near future. We also foresee that the implementation of QbD will lead to the development of more robust and efficient manufacturing processes and to a new generation of mAbs with increased clinical efficacy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Differential scanning calorimetry and fluorimetry measurements of monoclonal antibodies and reference proteins: Effect of scanning rate and dye selection

Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) were used to measure the transition temperatures of four proteins: RNase A, invertase, rituximab, and the NISTmAb (NIST Reference Material, RM 8671). The proteins were combined with several different fluorescent dyes for the DSF measurements. This study compares the results of DSC and DSF measurements of transition temperatures with different types of proteins, dye combinations, and thermal scan rates. As protein unfolding is often influenced by kinetic effects, we measured the transition temperatures of the proteins using DSC over a range of temperature scan rates and compared them to the data obtained from DSF over comparable temperature scan rates. The results when the proteins were combined with Sypro Orange^® and bis‐ANS for the DSF measurements had the best correlations with the transition temperatures determined by calorimetry. The scan rate was found to be an important variable when comparing results between DSC and DSF. The van't Hoff enthalpy changes for the transitions were calculated from the DSC data by using a non‐two‐state model and from the DSF values using a two‐state model. The calculated van't Hoff enthalpy changes did not show a good correlation between the two methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:677–686, 2017     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles

Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.     

Modified antigen-binding of human antibodies with glycosylation variations of the light chains produced in sugar-limited human hybridoma cultures

Summary We have characterized the effects of serum andN-acetylglucosamine in a glucose-deprived condition on the glycosylation of antibody light chains, as well as the resulting biological properties of those antibodies. We have chosen for our investigation the human hybridoma lines producing monoclonal antibodies reactive to lung adenocarcinoma. Each antibody possess aN-glycosylated carbohydrate chain in the hypervariable region of the light chains. When the cell lines were grown in the absence of glucose, variant light chains with varying molecular masses were found to be secreted. Analysis of these light chains produced in a glucose-deprived condition revealed that the changed molecular-mass of the variant light chains is due to different glycosylation. Addition ofN-acetylglucosamine or fetal calf serum to the glucose-free medium led to the creation of other light chains that exhibit increased antigen binding activity.     

Structural Alteration of Humic Acids by Pseudomonas spp. from Deep Terrestrial Subsurface Diatomite Formations in Northernmost Japan

Bacterial utilization of humic acids (HAs) was examined under aerobic conditions using Pseudomonas spp. from diatomite from a depth of 250 m below ground level in the Horonobe Underground Research Laboratory. HA decolorization and bacterial aggregation were observed during cultivation when an auxiliary carbon source was added. High-performance size-exclusion chromatography showed that high-molecular-weight HAs were produced. Fourier transform infrared spectroscopy indicated that carboxyl groups and polysaccharide-related substances in HAs were eliminated, while aliphatic structural units and amide groups were added to HAs. These results suggested that Pseudomonas spp. utilize and alter the molecular structure of HAs under aerobic conditions caused by the construction of underground facilities.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Moisture sorption characteristics and thermophysical properties of trehalose-PBS mixtures

The goal of the study was to quantify the thermophysical properties and the moisture sorption characteristics of the trehalose-PBS (phosphate-buffered saline) from the desiccation preservation perspective. A moisture sorption study was undertaken to determine the desorption isotherms of the trehalose-PBS mixtures. The Brunauer, Emmett, and Teller (BET)-equation and the Guggenheim, Anderson, and de Boer equation were used to quantify the desorption data. The glass transition temperature of the mixtures of trehalose-PBS, equilibrated at different relative humidities was studied using a differential scanning calorimeter. Fourier transform infrared spectroscopy was used to study the molecular interaction between the trehalose and PBS mixtures. The results showed that the addition of PBS to the trehalose mixture causes a shift from the type II isotherm to a type III isotherm (characterized by BET equation) which may have detrimental effect on cell desiccation. The results showed that an increase in PBS mass fraction in the trehalose-PBS mixture causes a decrease in the glass transition temperature (Tg) of the mixture and also a decrease in the hydrogen bonding capacity of the trehalose glasses. The addition of PBS to trehalose posed some challenges and should be subject to further optimization to use it in desiccation preservation of biologics.     

Simultaneous monitoring of oxidation,deamidation, isomerization,and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

Monoclonal antibodies are subjected to a wide variety of post-translational modifications (PTMs) that cause structural heterogeneity. Characterization and control of these modifications or quality attributes are critical to ensure antibody quality and to define any potential effects on the ultimate safety and potency of antibody therapeutics. The biopharmaceutical industry currently uses numerous tools to analyze these quality attributes individually, which requires substantial time and resources. Here, we report a simple and ultrafast bottom-up liquid chromatography-mass spectrometry (uLC-MS) method with 5 min tryptic digestion to simultaneously analyze multiple modifications, including oxidation, deamidation, isomerization, glycation, glycosylation, and N-terminal pyro-glutamate formation, which can occur during antibody production in mammalian cell culture, during purification and/or on storage. Compared to commonly used preparation procedures, this uLC-MS method eliminates assay artifacts of falsely-increased Met oxidation, Asp isomerization, and Asn deamidation, a problem associated with long digestion times in conventional LC-MS methods. This simple, low artifact multi-attribute uLC-MS method can be used to quickly and accurately analyze samples at any stage of antibody drug development, in particular for clone and media selection during cell culture development.     

Interaction of cyproheptadine hydrochloride with human serum albumin using spectroscopy and molecular modeling methods

The interaction between cyproheptadine hydrochloride (CYP) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT‐IR) and molecular modeling at a physiological pH (7.40). Fluorescence of HSA was quenched remarkably by CYP and the quenching mechanism was considered as static quenching since it formed a complex. The association constants K_a and number of binding sites n were calculated at different temperatures. According to Förster's theory of non‐radiation energy transfer, the distance r between donor (human serum albumin) and acceptor (cyproheptadine hydrochloride) was obtained. The effect of common ions on the binding constant was also investigated. The effect of CYP on the conformation of HSA was analyzed using FT‐IR, synchronous fluorescence spectroscopy and 3D fluorescence spectra. The thermodynamic parameters ΔH and ΔS were calculated to be ?14.37 kJ mol^?1 and 38.03 J mol^?1 K^?1, respectively, which suggested that hydrophobic forces played a major role in stabilizing the HSA‐CYP complex. In addition, examination of molecular modeling indicated that CYP could bind to site I of HSA and that hydrophobic interaction was the major acting force, which was in agreement with binding mode studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Preparation and preliminary application of monoclonal antibodies against Trichokirin-S1, a small ribosome-inactivating peptide from the seeds of Trichosanthes kirilowii

Trichokirin-S1,a small ribosome-inactivating peptide recently purified from the seeds ofTrichosanthes kirilowii,has potential clinical applications because of its small molecular mass.Two stablestrains of hybridomas (1F11 and 2A5) that can secrete highly specific monoclonal antibodies (mAbs) againstTrichokirin-S1 have been developed using the hybridoma technique.The isotypes of these two mAbs,1F11and 2A5,were determined to be IgG_(2a) and IgG_1,respectively.The affinity constants,which were measuredby non-competitive ELISA,were found to be 2.3×10~8 M~(-1) and 2.8×10~8 M~(-1),respectively.An immunoaffinitymethod using 2A5-coupled Sepharose 4B was successfully developed to purify Trichokirin-S1.These twoantibodies have also been used to detect Trichokirin-S1 in Western blot.     

Characterization of the co‐elution of host cell proteins with monoclonal antibodies during protein A purification

Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co‐purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D‐LC‐HDMS^E approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (～10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co‐purification with individual Fc and (Fab′)₂ antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708–717, 2016