分析单克隆抗体电荷异质性的成像毛细管等电聚焦电泳技术平台方法的建立

目的  采用成像毛细管等电聚焦电泳技术（imaged capillary isoelectric focusing, iCIEF）建立分析单克隆抗体（单抗）电荷异质性的平台方法,并用不同亚型单抗（IgG1、IgG2、IgG4）确认该平台方法的适用性。方法  优化该平台方法的部分参数,包括两性电解质和阴极稳定剂体积、聚焦时间和尿素浓度。采用3种亚型单抗（IgG1、IgG2、IgG4）对该平台方法的专属性、精密度、线性、准确度和耐用性进行验证。结果   对样品（单抗）的处理条件为：3 mol/L尿素-0.5%甲基纤维素溶液70 μl、两性电解质（pH3~10）4 μl、阴极稳定剂（500 mmol/L 精氨酸）2 μl、等电点 6.14和9.99 Marker 各2 μl,最终完成0.2 mg/ml单抗（样品）的制备。检测参数：预聚焦1 500 V、1 min,聚焦3 000 V、8 min。该平台方法的专属性良好,制剂缓冲液对检测无干扰。重复检测6份平行样品以及不同分析员于不同时间检测12份样品各成分含量的相对标准偏差均符合规定的要求。单抗（样品）终浓度为0.1～0.3 mg/ml时,主要和酸性成分的线性决定系数（R2）≥0.99,碱性成分的线性R2≥0.98。该平台方法检测样品各成分的准确度为92～105%。耐用性实验设计结果表明,两性电解质（pH3~10）体积和毛细管批次对该平台方法有显著影响。结论  建立的iCIEF平台方法分离度较高,精密度、准确度和耐用性良好,为单抗制品的电荷异质性表征和质量控制提供了更有效的工具。     

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.     

Investigation of Metal-Catalyzed Antibody Carbonylation With an Improved Protein Carbonylation Assay

Protein carbonylation is a posttranslational modification referring to the occurrence of aldehydes and ketones in proteins. The current understanding of how carbonylation, in particular, metal-catalyzed carbonylation, occurs in recombinant mAbs during production and storage is very limited. To facilitate investigations into mAb carbonylation, we developed a protein carbonylation assay with improved assay robustness and precision over the conventional assays. We applied this assay to investigate mAb carbonylation under production, storage, and stress conditions and showed that iron, hydrogen peroxide, and polysorbate 20 at pharmaceutically relevant levels critically influence the extent of mAb carbonylation. In addition, we found that while carbonylation correlates with mAb aggregation in several cases, carbonylation cannot be used as a general indicator for aggregation. Furthermore, we observed that mAb carbonylation level can decrease during storage, which indicates that carbonylation products may not be stable. Finally, we report for the first time a positive correlation between carbonylation and acidic charge heterogeneity of mAbs that underwent metal-catalyzed oxidation. This finding shows that the impact of protein carbonylation on product quality for mAbs is not limited to aggregation but also extends to charge heterogeneity.     

重组抗人表皮生长因子受体2单克隆抗体的电荷异质体分析

目的  探讨重组抗人表皮生长因子受体2（human epidermal growth factor receptor 2,Her2）单克隆抗体（单抗）的酸碱异质体对其生物活性和亲和力的影响。方法  分别用过氧化氢、肽-N-糖苷酶F、羧肽酶B对抗Her2单抗进行氧化、脱糖、酶切处理。用阳离子交换高效液相色谱法和毛细管等电聚焦电泳分析抗Her2单抗的电荷异质体,使用微量差示扫描荧光法分析抗Her2单抗的热稳定性,用细胞增殖抑制法和表面等离激元共振技术分析抗Her2单抗的亲和力。结果  脱糖处理抗Her2单抗的酸性电荷异质体增加了5.58%,与免疫球蛋白G Fc受体1（high affinity immunoglobulin gamma Fc receptor Ⅰ,FcGR1A）的亲和力明显减弱,亲和力常数为9.032×10^-8 mol/L。氧化和酶切处理抗Her2单抗的热稳定性降低,去折叠温度分别为63.6和59.5 ℃,均低于未处理抗Her2单抗（66.7 ℃）。结论  3种处理均会使抗Her2单抗产生电荷异质性,脱糖处理抗Her2单抗与FcGR1A的亲和力减弱,氧化和酶切处理抗Her2单抗的热稳定性降低。     

Characterization of Asparagine Deamidation and Aspartate Isomerization in Recombinant Human Interleukin-11

Purpose. The aim of this study was to investigate asparagine (Asn) deamidation and aspartate (Asp) isomerization and to measure the content of isoaspartate (isoAsp) in recombinant human interleukin-11 (rhIL-11). Methods. The rhIL-11 control and heat stressed samples were characterized with trypsin and endoproteinase Asp-N peptide mapping, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry (ESI MS) and capillary electrophoresis (CE). The total isoAsp content and bioactivity were also assessed. Results. Stress of rhIL11 at 30°C for 6 weeks in liquid resulted in significant isomerization of Asp⁴⁵ and Asp⁴⁷. Isomerization of Asp⁵¹ and deamidation of Asn⁴⁹ were also detected at low levels. The stressed rhIL-11 molecule contained 0.3 mol of isoAsp per mol of protein, compared to only 0.007 mol/mol of protein in the control. Conclusions. Asp and Asn residues, located in a loop structure of rhIL-11, undergo isoAsp formation under stressed conditions.     

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.     

Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development

The N-terminal glutamic acid (Glu) can be cyclized to form pyroglutamate (pGlu). Recent studies have suggested that N-terminal pGlu formation is an important posttranslational or co-translational event and is greatly facilitated by the enzyme glutaminyl cyclase, although the impact of the N-terminal cyclization on the potency and overall stability of mAbs is not been well known. Since most recombinant monoclonal antibodies (mAbs) contain glutamic acid and/or glutamine at their N-terminus, understanding the cyclization mechanisms may shed light on the factors that control the pGlu formation in therapeutic mAb development. Here, two mass spectrometry-based techniques were developed to investigate N-pyroglutamyl formation and the high conversion rate to pGlu at the N-terminus of the mAb was reported in the formulation development. The pGlu formation is favored at pH 4 and 8, but is less common at the neutral pH that is optimum for the enzymatic Glu conversion. These observations suggest that pGlu formation can proceed non-enzymatically at mild conditions and that this cyclization is not driven by glutaminyl cyclase in non-physiological conditions. We also calculate the half-lives of the N-terminal Glu at different pH and temperatures from the kinetics data, which would be very helpful for predicting pGlu formation and for selecting proper formulation and storage conditions.     

单抗药物的糖化修饰及其影响

目的：对单抗糖化修饰的产生、检测和其对单抗功能活性的影响进行综述,为行业提供参考。方法：通过文献检索,对国内外开展的单抗糖化研究进行梳理和汇总。结果与结论：单抗糖化主要发生在细胞发酵阶段,糖化的程度和速率受多种因素的影响,糖化修饰可以对单抗的异质性、功能活性以及免疫原性产生一定的影响。目前国内研发企业在单抗药学研究中通常忽视对糖化修饰的研究和评价,建议在单抗研发时应充分评估糖化的影响并确定是否为关键质量属性,并对其进行有效的控制和监测。     

Concentration Dependent Viscosity of Monoclonal Antibody Solutions: Explaining Experimental Behavior in Terms of Molecular Properties

Purpose

Early identification of monoclonal antibody candidates whose development, as high concentration (≥100 mg/mL) drug products, could prove challenging, due to high viscosity, can help define strategies for candidate engineering and selection.

Methods

Concentration dependent viscosities of 11 proprietary mAbs were measured. Sequence and structural features of the variable (Fv) regions were analyzed to understand viscosity behavior of the mAbs. Coarse-grained molecular simulations of two problematic mAbs were compared with that of a well behaved mAb.

Results

Net charge, ξ-potential and pI of Fv regions were found to correlate with viscosities of highly concentrated antibody solutions. Negative net charges on the Fv regions of two mAbs with poor viscosity behaviors facilitate attractive self-associations, causing them to diffuse slower than a well-behaved mAb with positive net charge on its Fv region. An empirically derived equation that connects aggregation propensity and pI of the Fv region with high concentration viscosity of the whole mAb was developed.

Conclusions

An Fv region-based qualitative screening profile was devised to flag mAb candidates whose development, as high concentration drug products, could prove challenging. This screen can facilitate developability risk assessment and mitigation strategies for antibody based therapeutics via rapid high throughput material-free screening.     

A novel method for quantitative measurement of a therapeutic monoclonal antibody in the presence of its target protein using enzymatic digestion

Over the past decades, the use of therapeutic monoclonal antibodies (mAbs) has become an important strategy in the treatment of various diseases. To enable pharmacokinetic (PK) assessment, specific immunoassays need to be developed to quantify mAbs in blood. In these assays, the presence of bound target protein can lead to severe underestimation of mAb concentration. Here we describe a novel approach for the quantification of total (free plus bound) human mAb concentration, in human and non-human primate serum, in the presence of a high level of target protein. The method is based on sample digestion with pepsin under optimized conditions to fully digest the target while keeping the mAb in the form of immunoreactive fragments. The quantification of mAb is then performed by ELISA without interference from the target. This method allows accurate quantification of as low as 50 ng/ml mAb in the presence of up to 100-fold target molar excess. Intra- and inter-run precision is better than 10%, and intra- and inter-run accuracy in the range of 89.3–106.7%. In conclusion, this general and simple approach allows the accurate and sensitive measurement of preclinical and clinical samples avoiding target interference.     

Matching pH values for antibody stabilization and crystallization suggest rationale for accelerated development of biotherapeutic drugs

Monoclonal antibodies (mAbs) are currently leading products in the global biopharmaceutical market. Multiple mAbs are in clinical development and novel biotherapeutic protein scaffolds, based on the canonical immunoglobulin G (IgG) fold, are emerging as treatment options for various medical conditions. However, fast approvals for biotherapeutics are challenging to achieve, because of difficult scientific development procedures and complex regulatory processes. Selecting molecular entities with superior physicochemical properties that proceed into clinical trials and the identification of stable formulations are crucial developability aspects. It is widely accepted that the solution pH has critical influences on both the protein's colloidal stability and its crystallization behavior. Furthermore, proteins usually crystallize best at solution conditions that enable high protein solubility, purity, stability, and monodispersity. Therefore, we hypothesize that the solution pH value is a central parameter that is linking together protein formulation, protein crystallization, and thermal protein stability. In order to experimentally test this hypothesis, we have investigated the effect of the solution pH on the thermal stabilities and crystallizabilities for three different mAbs. Combining biophysical measurements with high throughput protein (HTP) crystallization trials we observed a correlation in the buffer pH values for eminent mAb stability and successful crystallization. Specifically, differential scanning fluorimetry (DSF) was used to determine pH values that exert highest thermal mAb stabilities and additionally led to the identification of unfolding temperatures of individual mAb domains. Independently performed crystallization trials with the same mAbs resulted in their successful crystallization at pH values that displayed highest thermal stabilities. In summary, the presented results suggest a strategy how protein crystallization could be used as a screening method for the development of biotherapeutic protein formulations with improved in vitro stabilities.     

The Use of a GroEL-BLI Biosensor to Rapidly Assess Preaggregate Populations for Antibody Solutions Exhibiting Different Stability Profiles

An automated method using biotinylated GroEL-streptavidin biosensors with biolayer interferometry (GroEL-BLI) was evaluated to detect the formation of transiently formed, preaggregate species in various pharmaceutically relevant monoclonal antibody (mAb) samples. The relative aggregation propensity of various IgG1 and IgG4 mAbs was rank ordered using the GroEL-BLI biosensor method, and the least stable IgG4 mAb was subjected to different stresses including elevated temperatures, acidic pH, and addition of guanidine HCl. The GroEL-BLI biosensor detects mAb preaggregate formation mostly before, or sometimes concomitantly with, observing soluble aggregates and subvisible particles using size-exclusion chromatography and microflow imaging, respectively. A relatively unstable bispecific antibody (Bis-3) was shown to bind the GroEL biosensor even at low temperatures (25^°C). During thermal stress (50°C, 1 h), increased Bis-3 binding to GroEL-biosensors was observed prior to aggregation by size-exclusion chromatography or microflow imaging. Transmission electron microscopy analysis of Bis-3 preaggregate GroEL complexes revealed, in some cases, potential hydrophobic interaction sites between the Fc domain of the Bis-3 and GroEL protein. The automated BLI method not only enables detection of transiently formed preaggregate species that initiate protein aggregation pathways but also permits rapid mAb formulation stability assessments at low volumes and low protein concentrations.     

Oxidation and Deamidation of Monoclonal Antibody Products: Potential Impact on Stability,Biological Activity,and Efficacy

The role in human health of therapeutic proteins in general, and monoclonal antibodies (mAbs) in particular, has been significant and is continuously evolving. A considerable amount of time and resources are invested first in mAb product development and then in clinical examination of the product. Physical and chemical degradation can occur during manufacturing, processing, storage, handling, and administration. Therapeutic proteins may undergo various chemical degradation processes, including oxidation, deamidation, isomerization, hydrolysis, deglycosylation, racemization, disulfide bond breakage and formation, Maillard reaction, and β-elimination. Oxidation and deamidation are the most common chemical degradation processes of mAbs, which may result in changes in physical properties, such as hydrophobicity, charge, secondary or/and tertiary structure, and may lower the thermodynamic or kinetic barrier to unfold. This may predispose the product to aggregation and other chemical modifications, which can alter the binding affinity, half-life, and efficacy of the product. This review summarizes major findings from the past decade on the impact of oxidation and deamidation on the stability, biological activity, and efficacy of mAb products. Mechanisms of action, influencing factors, characterization tools, clinical impact, and risk mitigation strategies have been addressed.     

Differential tumor cell targeting of anti-HER2 (Herceptin) and anti-CD20 (Mabthera) coupled nanoparticles

Two types of antibody-labeled nanoparticles (mAb-NPs) were prepared with the aim to achieve specific tumor targeting. Anti-HER2 and anti-CD20 monoclonal antibodies (mAb) were used as model ligands. Small poly(dl-lactic acid) nanoparticles (PLA NPs) with a mean size of about 170 nm were prepared by the salting out method. Thereafter, the coating of PLA NPs with mAbs was performed in two steps. First, thiol groups (-SH) were introduced on the surface of PLA-NPs by a two-step carbodiimide reaction. The number of -SH groups on the surface of NPs increased from 150 to 400 mmol-SH/mol PLA when cystamine concentrations of 25-1518 mol cystamine/mol PLA were used during the thiolation reaction. In the second step, covalent coupling of antibodies to thiolated NPs (NPs-SH) was obtained via a bifunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (sulfo-MBS). For both mAbs anti-HER2 and anti-CD20, respectively, the number of -SH functions on the NPs had no influence on the amount of mAb coupled to the NPs. Approximately, 295 anti-HER2 and 557 anti-CD20 molecules, respectively, were covalently coupled per nanoparticle. The NPs size after the coupling reactions was about 250 nm. The specific interaction between tumor cells and mAb-NPs was determined by confocal microscopy using two cell lines: SKOV-3 human ovarian cancer cells (overexpressing HER2) and Daudi lymphoma cells (overexpressing CD20). The results showed the selective targeting of mAb-NPs to tumor cells overexpressing the specific antigen. While anti-CD20 labeled NPs (anti-CD20 NPs) bound to and remained at the cellular surface, anti-HER2 labeled NPs (anti-HER2 NPs) were efficiently internalized. The mAb-NPs represent a promising approach to improve the efficacy of NPs in active targeting for cancer therapy while the choice of the antibody-target system defines the fate of the mAb-NPs after their binding to the cells.     

Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab.     

Isoaspartate in peptides and proteins: formation, significance, and analysis

Formation of isoaspartyl peptide bonds (isoAsp) is one of the most common forms of non-enzymatic degradation of peptides and proteins under mild conditions. IsoAsp arises when certain Asn-Xaa and Asp-Xaa sites undergo a spontaneous intramolecular rearrangement to form a succinimide which subsequently hydrolyzes to generate a mixture of isoAsp-Xaa and Asp-Xaa linkages in a ratio of approximately 2:1. This pathway is responsible for the much greater susceptibility of asparagine, compared with glutamine, to deamidation at neutral and alkaline pH. Rearrangement occurs most readily at Asn-Gly, Asn-Ser, and Asp-Gly sequences where the local polypeptide chain flexibility is high. Formation of isoAsp can decrease the biological activity of a protein pharmaceutical, alter its susceptibility to proteolytic degradation, and elicit autoimmunity. The enzyme protein L-isoaspartyl methyltransferase can be used to measure isoAsp sites in the low pmol range with or without the use of radioisotopes.     

Epitopic characterization of the human wild-type and mutant ras proteins using membrane-bound peptides

Overlapping octapeptides encompassing the entire sequences of the human oncogene products Ha-ras, K-ras and N-ras protein were synthesized as spots on polypropylene membrane sheets. The binding of anti-row protein monoclonal antibodies (mAbs) to the membrane-bound peptides was assessed using an enzyme-linked immunosorbent assay. Epitopes of 10 of 18 mAbs to the human ras proteins were mapped and identified by this procedure. The epitopes of nine of the mAbs are within residues 28-39 in the constant domain common to the three ras proteins, whereas the epitope of the tenth (mAb 21) spans residues 136-144 in Ha-ras. The minimal lengths of epitopes of all ten of the mAbs were further precisely mapped using peptides of varying length, and the tolerance for mAb binding of mutated epitopes was determined by systematically replacing each residue in the epitope with each of the 20 common amino acids. The results show that most of these mAbs have essentially the same binding specificity, namely for the sequence YDPT (residues 32–35) or for slightly longer sequences containing these residues. This site is in the switch 1 region (residues 32-38) in the ras effector loop, indicating that some of the same residues important for the interaction of ras with other proteins (GTPase-activating protein, neurofibromin or raf) are highly antigenic. In addition, we investigated epitopes and specificity of five mAbs against the activated human ras proteins by the same procedure. The information gained from this study should be useful both for study of the complicated functions of ras proteins and for clinical detection of ras oncogenes in human tumor cells.     

Characterization, epitope identification and mechanisms of the anti-septic capacity of monoclonal antibodies against macrophage migration inhibitory factor

Sepsis is characterized by uncontrolled inflammatory responses. Macrophage migration inhibitory factor (MIF) has been shown to play an important role in the progression of sepsis thus is a potential therapeutic target. The aim of this study is to produce IgG anti-MIF monoclonal antibodies (mAbs) with anti-septic abilities in vivo and to determine mechanisms of their function. We generated 8 IgG anti-MIF mAbs with high specificity and 3 of them showed potent protective abilities in murine lethal peritonitis induced by cecal ligation and puncture (CLP). One anti-MIF mAb, F11, showed 100% protection within 72 h after sepsis induction and 72% mice treated with this mAb survived up to 84 h with reduced lung and kidney pathology. F11 treatment also reduced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in septic mice. We further found that all 8 anti-MIF mAbs recognized the same epitope located in the amino acid residue 1-20 region of the N terminus of the MIF protein. Three of the mAbs, F11 in particular, inhibited tautomerase activity in association with their protective effect on CLP mice. Thus, we have produced anti-MIF mAbs that protected mice from CLP-induced sepsis by recognizing the same epitope domains in MIF. These mAbs are promising candidates for further development of therapeutics against inflammatory diseases.     

Laser-induced breakdown detection of temperature-ramp generated aggregates of therapeutic monoclonal antibody

The detection and characterization of protein aggregation is essential during development and quality control of therapeutic proteins, as aggregates are typically inactive and may trigger anti-drug-antibody formation in patients. Especially large multi-domain molecules, such as the important class of therapeutic monoclonal antibodies (mAbs), can form various aggregates that differ in size and morphology. Although particle analysis advanced over the recent years, new techniques and orthogonal methods are highly valued. To our knowledge, the physical principle of laser-induced breakdown detection (LIBD) was not yet applied to sense aggregates in therapeutic protein formulations. We established a LIBD setup to monitor the temperature-induced aggregation of a mAb. The obtained temperature of aggregation was in good agreement with the results from previously published temperature-ramped turbidity and dynamic light scattering measurements. This study demonstrates the promising applicability of LIBD to investigate aggregates from therapeutic proteins. The technique is also adaptive to online detection and size determination, and offers interesting opportunities for morphologic characterization of protein particles and impurities, which will be part of future studies.     

Determination of Interaction Parameters for Reversibly Self-Associating Antibodies: A Comparative Analysis

Monoclonal antibodies (mAbs) represent a major class of biotherapeutics and are the fastest growing category of biologic drugs on the market. However, mAb development and formulation are often impeded by reversible self-association (RSA), defined as the dynamic exchange of monomers with native-state oligomers. Here, we present a comparative analysis of the self-association properties for 5 IgG mAbs, under matched conditions and using orthogonal methods. Concentration-dependent dynamic light scattering and sedimentation velocity studies revealed that the majority of mAbs examined exhibited weak to moderate RSA. However, because these studies were carried out at mAb concentrations in the mg/mL range, we also observed significant nonideality. Noting that nonideality frequently masks RSA and vice versa, we conducted direct boundary fitting of the sedimentation velocity data to determine stoichiometric binding models, interaction affinities, and nonideality terms for each mAb. These analyses revealed equilibrium constants from micromolar to millimolar and stoichiometric models from monomer-dimer to isodesmic. Moreover, even for those mAbs described by identical models, we observed distinct kinetics of self-association. The accuracy of the models and their corresponding equilibrium constants were addressed using sedimentation equilibrium and simulations. Overall, these results serve as the starting point for the comparative dissection of RSA mechanisms in therapeutic mAbs.