Impact of mammalian cell culture conditions on monoclonal antibody charge heterogeneity: an accessory monitoring tool for process development

Recombinant monoclonal antibodies are predominantly produced in mammalian cell culture bioprocesses. Post-translational modifications affect the micro-heterogeneity of the product and thereby influence important quality attributes, such as stability, solubility, pharmacodynamics and pharmacokinetics. The analysis of the surface charge distribution of monoclonal antibodies provides aggregated information about these modifications. In this work, we established a direct injection pH gradient cation exchange chromatography method, which determines charge heterogeneity from cell culture supernatant without any purification steps. This tool was further applied to monitor processes that were performed under certain process conditions. Concretely, we were able to provide insights into charge variant formation during a fed-batch process of a Chinese hamster ovary cell culture, in turn producing a monoclonal antibody under varying temperatures and glucose feed strategies. Glucose concentration impacted the total emergence of acidic variants, whereas the variation of basic species was mainly dependent on process temperature. The formation rates of acidic species were described with a second-order reaction, where a temperature increase favored the conversion. This platform method will aid as a sophisticated optimization tool for mammalian cell culture processes. It provides a quality fingerprint for the produced mAb, which can be tested, compared to the desired target and confirmed early in the process chain.

     

Kinetic modeling as a tool to understand the influence of cell culture process parameters on the glycation of monoclonal antibody biotherapeutics

Glycation, the nonenzymatic reaction between the reducing sugar glucose and the primary amine residues on amino acid side chains, commonly occurs in the cell culture supernatant during production of therapeutic monoclonal antibodies (mAbs). While glycation has the potential to impact efficacy and pharmacokinetic properties for mAbs, the most common undesirable impact of glycation is on the distribution of charged species, often a release specification for commercial processes. Existing empirical approaches are usually insufficient to rationalize the effects of cell line and process changes on glycation. To address this gap, we developed a kinetic model for estimating mAb glycation levels during the cell culture process. The rate constant for glycation, including temperature and pH dependence, was estimated by fitting the kinetic model to time-course glycation data from bioreactors operated at different process settings that yielded a wide range of glycation values. The parameter values were further validated by independently estimating glycation rate constants using cell-free incubation studies at various temperatures. The model was applied to another mAb, by re-estimating the activation energy to account for effect of a glycation “hotspot”. The model was further utilized to study the role of temperature shift as an approach to reduce glycation levels in the manufacturing process for mAb2. While a downshift in temperature resulted in lowering of glycation levels for mAb2, the model helped elucidate that this effect was caused due to contribution from changes in glucose consumption, mAb secretion and temperature, instead of a direct impact of temperature alone on the kinetic rate of glycation.     

Elucidating the effects of pH shift on IgG1 monoclonal antibody acidic charge variant levels in Chinese hamster ovary cell cultures

Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies’ properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Oxidative stress on the astrocytes in culture derived from a senescence accelerated mouse strain

Astrocytes are one of the predominant glial cell types in the adult central nervous system functioning as both supportive and metabolic cells for the brain. Our objective in this experiment is to study the direct effects of hydrogen peroxide induced oxidative stress on astrocytes in culture. These astrocytes were derived from both an aged mouse strain (P8) and a matched control strain (R1). The astrocytes for both the P8 and R1 strains were treated with increasing concentrations of hydrogen peroxide. Our results showed that the oxidative stress had a similar effect in both strains of astrocytes; decreases in 3-(4,5-dimethylthiazol-2-yl)-2,2-diphenyltetrazolium bromide (MTT) and glial fibrillary acidic protein (GFAP) levels, and increases in terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, lactate dehydrogenase (LDH) staining, and superoxide dismutase (SOD), caspase-3 and B-cell lymphoma 2-associated protein X (bax) levels. At a hydrogen peroxide concentration of 400 microM , the differences of the above parameters between P8 cultures and R1 cultures were statistically significant (p<0.05). This strongly suggested that astrocytes derived from P8 and R1 strains reacted to oxidative stress with similar mechanisms and consequences. However, the mechanisms were not able to compensate for the oxidative stress in the P8 strain at a hydrogen peroxide concentration of 400 microM. The inability of the P8 astrocytes to counteract the oxidative stress might lead to inadequate protection from neuronal loss possibly resulting in significantly more astrocytic death. Our results suggested that the changes of astrocytes in peroxide detoxification may play a role in aging of the central nervous system, and further aging studies should examine the oxidative status of the samples.     

Characterization of a unique IgG1 mAb CEX profile by limited Lys-C proteolysis/CEX separation coupled with mass spectrometry and structural analysis

The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.     

Host cell protein dynamics in the supernatant of a mAb producing CHO cell line

The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile.     

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%–17%), main (18%–23%), and basic (59%–68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.     

Ex vivo detection of histone H1 modified with advanced glycation end products

A number of oxidative stress agents cause DNA and protein damage, which may compromise genomic integrity. Whereas oxidant-induced DNA damage has been extensively studied, much less is known concerning the occurrence and fate of nuclear protein damage, particularly of proteins involved in the regulation and maintenance of chromatin structure. Protein damage may be caused by the formation of reactive carbonyl species such as glyoxal, which forms after lipid peroxide degradation. It may also result from degradation of early protein glycation adducts and from methylglyoxal, formed in the process of glycolytic intermediate degradation. Major adducts indicative of protein damage include the advanced glycation end product (AGE) carboxymethyllysine (CML) and argpyrimidine protein adducts. Thus, the formation of CML and argpyrimidine protein adducts represents potential biomarkers for nuclear protein damage deriving from a variety of sources. The purpose of this study was to identify and quantify AGE adducts formed in vivo in a nuclear protein, specifically histone H1, using CML and argpyrimidine as biomarkers. Histone H1 was isolated from calf thymus collected immediately after slaughter under conditions designed to minimize AGE formation before isolation. Using antibodies directed against oxidative protein adducts, we identified CML, argpyrimidine, and protein crosslinks present in the freshly isolated histone H1. Detailed mass spectroscopy analysis of histone H1 revealed the presence of two specific lysine residues modified by CML adducts. Our results strongly suggest that glycation of important nuclear protein targets such as histone H1 occurs in vivo and that these oxidative changes may alter chromatin structure, ultimately contributing to chronic changes associated with aging and diseases such as diabetes.     

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short term and prolonged oxidative stress

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short-term and prolonged oxidative stress was studied. An increase in the activity of intracellular catalase in fungal cells after short-term exposure to hydrogen peroxide was shown. Activation of fungal cells induced by H2O2 depends on H2O2 concentration, time of exposure, and the growth phase of the fungus. Variants of P. piceum F-648 that produced two forms of extracellular catalase with different catalytic properties were obtained due to prolonged adaptation to H2O2. Catalase with low affinity for substrate was produced predominantly by the parent culture and variant 3; however, a high substrate affinity of catalase was observed in variant 5. Variant 5 of P. peniceum F-648 displayed a high catalytic activity and operational stability of catalase in the presence of phosphate ions and the concentration of substrate less than 30 mM at pH more than 7.     

Improving product quality and productivity of bispecific molecules through the application of continuous perfusion principles

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12-day fed-batch process and an approximately 40-day high-density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15-fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed-batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity-capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity-determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds.     

Modes of shedding of glycosphingolipids from mouse lymphoma cells

To characterize the process by which glycolipids are shed from cell membranes, the cellular and supernatant glycolipids were compared from a variant of the mouse lymphoma L5178Y which had been selected for strong expression of the neutral glycolipid gangliotriaosylceramide (GgOse3Cer). This glycolipid was present in three forms which differed in their fatty acid composition. Whereas the major cell-associated form of GgOse3Cer contained C24 fatty acids, the predominant form shed into the culture supernatant contained C16 fatty acids. Ultracentrifugation of the culture medium yielded a pellet with a GgOse3Cer profile similar to that of the cells and a supernatant enriched in the C16 fatty acid form. Gel filtration of the culture medium revealed two GgOse3Cer-containing pools. The first was excluded from Sepharose CL-2B and had a GgOse3Cer profile similar to that of the cells, while the second migrated with proteins in the range of 25,000-500,000 daltons and was enriched in the C16 fatty acid form. These results suggest two forms in which glycolipids are released from cell membranes. The first is in a large complex, possibly a membrane vesicle, which retains the glycolipid profile of the membrane of intact cells while the second form appears to result from the preferential release of particular glycolipid components.     

Oxidative stress response in an anaerobe, Bacteroides fragilis: a role for catalase in protection against hydrogen peroxide.

Survival of Bacteroides fragilis in the presence of oxygen was dependent on the ability of bacteria to synthesize new proteins, as determined by the inhibition of protein synthesis after oxygen exposure. The B. fragilis protein profile was significantly altered after either a shift from anaerobic to aerobic conditions with or without paraquat or the addition of exogenous hydrogen peroxide. As determined by autoradiography after two-dimensional gel electrophoresis, approximately 28 newly synthesized proteins were detected in response to oxidative conditions. These proteins were found to have a broad range of pI values (from 5.1 to 7.2) and molecular weights (from 12,000 to 79,000). The hydrogen peroxide- and paraquat-inducible responses were similar but not identical to that induced by oxygen as seen by two-dimensional gel protein profile. Eleven of the oxidative response proteins were closely related, with pI values and molecular weights from 5.1 to 5.8 and from 17,000 to 23,000, respectively. As a first step to understanding the resistance to oxygen, a catalase-deficient mutant was constructed by allelic gene exchange. The katB mutant was found to be more sensitive to the lethal effects of hydrogen peroxide than was the parent strain when the ferrous iron chelator bipyridyl was added to culture media. This suggests that the presence of ferrous iron in anaerobic culture media exacerbates the toxicity of hydrogen peroxide and that the presence of a functional catalase is important for survival in the presence of hydrogen peroxide. Further, the treatment of cultures with a sublethal concentration of hydrogen peroxide was necessary to induce resistance to higher concentrations of hydrogen peroxide in the parent strain, suggesting that this was an inducible response. This was confirmed when the bacterial culture, treated with chloramphenicol before the cells were exposed to a sublethal concentration of peroxide, completely lost viability. In contrast, cell viability was greatly preserved when protein synthesis inhibition occurred after peroxide induction. Complementation of catalase activity in the mutant restored the ability of the mutant strain to survive in the presence of hydrogen peroxide, showing that the catalase (KatB) may play a role in oxidative stress resistance in aerotolerant anaerobic bacteria.     

Quantitative analysis of glycation and its impact on antigen binding

Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.     

Effect of ambient light on monoclonal antibody product quality during small‐scale mammalian cell culture process in clear glass bioreactors

During a small‐scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small‐scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:562–570, 2014     

Preferential precipitation of acidic variants from monoclonal antibody pools

Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variants in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein, and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs.     

Reactive oxygen species in in vitro pesticide-induced neuronal cell (SH-SY5Y) cytotoxicity: role of NFkappaB and caspase-3

Oxidative stress has been implicated in pesticide-induced neurotoxicity, based on its role in the cascade of biochemical changes that lead to dopaminergic neuronal cell death. We have, therefore, examined the role of oxidative stress caused by the pesticides endosulfan and zineb in human neuroblastoma cells (SH-SY5Y) in culture. Upon treatment with 50-200 microM concentrations of either of these pesticides, SH-SY5Y cells generated both superoxide anion and hydrogen peroxide in a dose-and time-dependent manner. Mixtures of the pesticides significantly enhanced the production of these reactive oxygen species compared to individual pesticide exposures. Pesticide treatment decreased superoxide dismutase, glutathione peroxidase, and catalase activities in SH-SY5Y cells. Additionally, these pesticides induced lipid peroxide (thiobarbituric acid reactive products) formation in these cells. While both pesticides individually (at 100 microM) increased caspase-3 activity, cells exposed to a mixture of the pesticides exhibited significantly low levels of this enzyme, probably due to excessive necrotic cell death. Furthermore, exposure to these pesticides increased nuclear NFkappaB activity. Taken together, these findings suggest that the cytotoxicity of endosulfan and zineb, both individually and in mixtures may, at least in part, be associated with the generation of reactive oxygen species with concomitant increased expression of NFkappaB.     

Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies

The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.     

Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies

The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.