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BACKGROUND In mammalian cell culture processes, viable cell density ( VCD) is one of the most crucial parameters for monitoring of the current status of cell growth and protein production. Among the various real-time monitoring tools available for VCD in Chinese hamster ovary ( CHO) cell culture, dielectric spectroscopy is one of the more reliable due to its simplicity, robustness and accuracy. These attributes enable efficient monitoring and control of the corresponding processes. However, one of the major drawbacks of dielectric spectroscopy is frequent deterioration of accuracy during the late-growth phase of CHO cell culture. This can be caused by abrupt changes in the cell physiology, as well as environmental conditions. RESULTS In this study, various kinds of chemometric tools were employed to understand the spectral dynamics of dielectric spectroscopy during extended CHO cell culture, including different cell growth phases. Then, the performance of different multivariate regression models were compared with conventional Cole-Cole equation-based approaches. The models were compared in their ability to estimate VCD in CHO cell culture from dielectric spectra from several batches of bioreactor operations. Multivariate analysis of dielectric spectral datasets clearly revealed the time-varying evolution of spectral dynamics during the transition between cell growth phases. The best prediction performance from the dielectric spectroscopy was realized using a regression model based on locally-weighted partial least squares ( LWPLS). This estimation model best compensated for spectral dynamics observed during the different cell growth phases. CONCLUSION Dielectric spectroscopy combined with LWPLS allows achievement of optimal and stable estimation of VCD during CHO cell culture, regardless of inherent differences in cell growth phases. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

ABSTRACT Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)-type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. Biotechnol. Bioeng. 2014;111: 95-103. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Separation of viruses and other contaminants from protein therapeutics using anion exchange membrane adsorbers is a successful new approach to viral clearance; however, the fundamental phenomena that control performance are not well understood. For example, the kinetics of adsorption to the anion exchange surface may limit clearance, but has yet to be characterized experimentally and mathematically. In the present study, surface plasmon resonance was used to determine the adsorption kinetics for five large biological molecules: phage PP7, phage ΦX174, phage PR772, thyroglobulin, and DNA. Rate constants were incorporated into a kinetic model of chromatography to illustrate the impact on performance. [ABSTRACT FROM AUTHOR]

The major histocompatibility complex (MHC) of the BALB/c mouse contains three genes encoding classical class I molecules, as well as at least 32 nonclassical class I genes. Although much is known about the genes encoding the classical class I molecules, the majority of the nonclassical genes have not been characterized. This report describes a newly identified nonclassical class I gene, Thy19.4, which contains an open reading frame and resembles in several regards the genes encoding classical class I molecules. The similarities include shared amino acid sequence motifs which suggest that the putative Thy19.4 molecule may assume a tertiary structure similar to that of the classical class I molecules, as well as widespread transcription in a variety of tissues. However, unlike the classical class I genes, the Thy19.4 gene maps approximately 1 cm distal to the Tla region of the MHC, in the same region as the gene encoding the Hmt element of the maternally transmitted antigen. [ABSTRACT FROM AUTHOR]

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2, D3, and D4, in addition to H-2Dand H-2L. We have determined the DNA sequence of the D2gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2gene is similar to other class I genes. It also contains similar 5′ regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2 molecule, we generated an exon-shuffled construct containing the promoter and exons 1-3, encoding the signal peptide, α1, and α2 external domains of the D2gene linked to exons 4-8, encoding the α3, transmembrane and cytoplasmic domains, of the H-2Dgene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the α3 domain of H-2D, as well as by other class I-specific mAbs. Although D2 is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene. [ABSTRACT FROM AUTHOR]

Abstract: The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines. ES-DMA is a rapid particle sizing method capable of characterizing certain aspects of the structure (such as capsid proteins) and obtaining complete size distributions of viruses and virus-like particles. It was shown that ES-DMA can distinguish intact virus particles from degraded particles and measure the concentration of virus particles when calibrated with nanoparticles of known concentration. The technique has a measurement uncertainty of ≈20%, is linear over nearly 3 orders of magnitude, and has a lower limit of detection of ≈109 particles/mL. This quantitative assay was demonstrated for non-enveloped viruses. It is expected that ES-DMA will be a useful method for applications involving production and quality control of vaccines and gene therapy vectors for human use. [Copyright &y& Elsevier]

Abstract: Mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are known to be occasional microbial contaminants of cell cultures that produce biologics. This presents a serious concern regarding the risk of mycoplasma contamination for research laboratories and commercial facilities developing and manufacturing cell-derived biological and biopharmaceutical products for therapeutic use. Potential undetected contamination of these products or process intermediates with mycoplasmas represents a potential safety risk for patients and a business risk for producers of biopharmaceuticals. To minimize these risks, monitoring for adventitious agents, such as viruses and mycoplasmas, is performed during the manufacture of biologics produced in cell culture substrates. The “gold standard” microbiological assay, currently recommended by the USP, EP, JP and the US FDA, for the mycoplasma testing of biologics, involves the culture of viable mycoplasmas in broth, agar plates and indicator cells. Although the procedure enables highly efficient mycoplasma detection in cell substrates and cell-derived products, the overall testing strategy is time consuming (a minimum of 28 days) and requires skilled interpretation of the results. The long time period required for these conventional assays does not permit their use for products with short shelf-lives or for timely ’go/no-go’ decisions during routine in-process testing. PCR methodology has existed for decades, however PCR based and other alternative methods for mycoplasma detection have only recently been considered for application to biologics manufacture. The application of alternative nucleic acid-based, enzyme-based and/or recombinant cell-culture methods, particularly in combination with efficient sample preparation procedures, could provide advantages over conventional microbiological methods in terms of analytical throughput, simplicity, and turnaround time. However, a challenge to the application of alternative methods for detection of mycoplasmas remains whether these alternative methods can provide a limit of detection comparable or superior to those of the culture methods. An additional challenge is that nucleic acid amplification technique (NAT) methods do not allow for accurate discrimination between viable and non-viable mycoplasma contaminants, which might lead to false-positive results (e.g. from inactivated raw materials, etc.). Our review provides an overview of these alternative methods and discusses the pros and cons of their application for the testing of mycoplasmas in biologics and cell substrates. [Copyright &y& Elsevier]

The presence of aggregates in monoclonal antibody (mAb) drug product (DP) formulations can present product quality challenges. Here we show that use of High Performance Size Exclusion Chromatography (HP-SEC), in conjunction with high-throughput dynamic light scattering (HT-DLS) analyses of mAb DPs can be a useful strategy to determine monomer content and the presence of aggregates under simulated stress conditions. This analytical approach was used to evaluate four commercially available mAb DPs under different conditions i.e.; original formulations, diluted, and thermo-mechanical stressed condition. Due to particle size limitations of HP-SEC columns, resulting in particles accumulating in the column frits prior to reaching the detector for analysis, there is a possibility that large mAb aggregates may not be detected. Both HP-SEC and HT-DLS were able to detect and resolve the mAb monomer (~10–12 nm) of the DPs in their recommended storage conditions. However, the ability to detect large aggregates (>40 nm) by both analytical methods differed, and HT-DLS was able to detect aggregates between 60 nm and 1400 nm under stress conditions. Our data indicates that HP-SEC, in conjunction with HT-DLS, may be beneficial to detect both mAb DP monomer content and multiple aggregate species (1–1000 nm) in the submicron size range. [ABSTRACT FROM AUTHOR]

There is a trend across the pharmaceutical sector toward process intensification and continuous manufacturing to produce small-molecule drugs or biotechnology products. For biotechnology products, advancing the manufacturing technology behind upstream and downstream processes has the potential to reduce product shortages and variability, allow for production flexibility, simplify scale-up procedures, improve product quality, reduce facility footprints, increase productivity, and reduce production costs. On the upstream side of biotechnology manufacturing, continuous perfusion cell cultures are fairly well established. However, truly integrated continuous biomanufacturing requires the uninterrupted connection of continuous unit operations (upstream and downstream) with no isolated intermediate or hold steps occurring between them. This work examines the current scientific and regulatory landscape surrounding the implementation of integrated continuous biomanufacturing. Highlights There is a trend across the pharmaceutical sector toward process intensification and continuous manufacturing to produce small-molecule drugs or biotechnology products. For biotechnology products, advancing the manufacturing technology behind upstream and downstream processes has the potential to reduce product shortages and variability, allow for production flexibility, simplify scale-up procedures, improve product quality, reduce facility footprints, increase productivity, and reduce production costs. Nevertheless, some scientific and regulatory challenges still exist in implementing integrated continuous biomanufacturing. [ABSTRACT FROM AUTHOR]

Highlights • An ICP-MS methodology to determine bulk, minor and trace metal content (0.05 ppb–500 ppb) of mammalian cell culture media. • An advanced autodilution platform for autocalibration and quality control to enhance throughput. • Application of this method to demonstrate the affected homogeneity of a model therapeutic due to spikes in the concentration of copper and iron in parallel bioreactors. Abstract Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced. [ABSTRACT FROM AUTHOR]

Stability of therapeutic proteins (TPs) is a critical quality attribute that impacts both safety and efficacy of the drug. Size stability is routinely performed during and after biomanufacturing. Dynamic light scattering (DLS) is a commonly used technique to characterize hydrodynamic size of the TPs. Herein, we have developed a novel method to evaluate in-use and thermal stress stability of TPs using algorithm-driven high-throughput DLS. Five marketed TPs were tested under the guidance of customized algorithms. The TPs were evaluated at relevant temperature conditions as well as under dilution and thermal stress for size stability. We found that the TPs were stable under the in-use conditions tested; however, sample loss due to evaporation can lead to large protein aggregates. A combined assessment of autocorrelation function and photos of sample well could be useful in formulation screening. Dilution of TPs also has an impact on the hydrodynamic size. Thermal stress experiments showed the importance of using different data processing methods to access size distribution. Polydispersity index was useful in evaluating sample heterogeneity. Herein, we show that algorithm-driven high-throughput DLS can provide additional supportive information during and after biomanufacturing and the potential to be used in a quality control environment. [ABSTRACT FROM AUTHOR]

Bacteriophage binding mechanisms to multi-modal anion exchange resin may include both anion exchange and hydrophobic interactions, or the mechanism can be dominated by a single moiety. However, previous studies have reported binding mechanisms defined for simple solutions containing only buffer and a surrogate viral spike (i.e. bacteriophage ΦX174, PR772, and PP7). We employed phage spiked in-process monoclonal antibody (mAb) pools to model binding under bioprocessing conditions. These experiments allow the individual contributions of the mAb, in-process impurities, and buffer composition on mechanistic removal of phages to be studied. PP7 and PR772 use synergetic binding by the positively charged quaternary amine and the hydrophobic aromatic phenyl group to bind multi-modal resin. ΦX174′s binding mechanism remains inconclusive due to operating conditions. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module. [ABSTRACT FROM AUTHOR]

A unique “design space (DSp) exploration strategy,” defined as a function of four key scenarios, was successfully integrated and validated to enhance the DSp building exercise, by increasing the accuracy of analyses and interpretation of processed data. The four key scenarios, defining the strategy, were based on cumulative analyses of individual models developed for the Critical Quality Attributes (23 Glycan Profiles) considered for the study. The analyses of the CQA estimates and model performances were interpreted as (1) Inside Specification/Significant Model (2) Inside Specification/Non-significant Model (3) Outside Specification/Significant Model (4) Outside Specification/Non-significant Model. Each scenario was defined and illustrated through individual models of CQA aligning the description. The R 2 , Q 2 , Model Validity and Model Reproducibility estimates of G2, G2FaGbGN, G0 and G2FaG2, respectively, signified the four scenarios stated above. Through further optimizations, including the estimation of Edge of Failure and Set Point Analysis, wider and accurate DSps were created for each scenario, establishing critical functional relationship between Critical Process Parameters (CPPs) and Critical Quality Attributes (CQAs). A DSp provides the optimal region for systematic evaluation, mechanistic understanding and refining of a QbD approach. DSp exploration strategy will aid the critical process of consistently and reproducibly achieving predefined quality of a product throughout its lifecycle. [ABSTRACT FROM AUTHOR]

Monoclonal antibody (mAb) drugs constitute the largest class of protein therapeutics currently on the market. Correctly folded protein higher order structure (HOS), including quinary structure, is crucial for mAb drug quality. The quinary structure is defined as the association of quaternary structures ( e.g. , oligomerized mAb). Here, several commonly available analytical methods, i.e. , size-exclusion-chromatography (SEC) FPLC, multi-angle light scattering (MALS), circular dichroism (CD), NMR and multivariate analysis, were combined and modified to yield a complete profile of HOS and comparable metrics. Rituximab and infliximab were chosen for method evaluation because both IgG1 molecules are known to be homologous in sequence, superimposable in Fab crystal structure and identical in Fc structure. However, herein the two are identified to be significantly different in quinary structure in addition to minor secondary structure differences. All data collectively showed rituximab was mostly monomeric while infliximab was in mono-oligomer equilibrium driven by its Fab fragment. The quinary structure differences were qualitatively inferred from the less used but more reproducible dilution-injection-SEC-FPLC curve method. Quantitative principal component analysis (PCA) was performed on NMR spectra of either the intact or the in-situ enzymatic-digested mAb samples. The cleavage reactions happened directly in NMR tubes without further separation, which greatly enhanced NMR spectra quality and resulted in larger inter- and intra-lot variations based on PCA. The new in-situ enzymatic digestion method holds potential in identifying structural differences on larger therapeutic molecules using NMR. [ABSTRACT FROM AUTHOR]

The objective of the study was to analyze the effect of controlled and uncontrolled freezing step of a lyophilization process on the extent of non-enzymatic glycation and aggregation of an IgG1 formulation at two concentrations (1 mg/ml and 20 mg/ml). The degree of glycation (%) was determined through boronate affinity chromatography and its effect on the formation of soluble aggregates and higher molecular weight species was studied using dynamic light scattering (DLS) and size exclusion chromatography with multi-angle light scattering (SEC-MALS). The effect of non-enzymatic glycation on the secondary structure of the formulations was also studied using circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. Results indicated that controlled nucleation yielded higher residual moisture contents and significantly lower specific surface areas for the two monoclonal antibody concentrations compared with uncontrolled nucleation cycle ( p < 0.05). For the two concentrations, uncontrolled nucleation resulted in significantly higher levels of glycation compared with controlled nucleation samples ( p < 0.05). Further, it was observed that higher storage temperatures (25 °C/60% RH) versus 5 °C resulted in higher glycation. Even though SEC-MALS analyses of the low concentrated formulations did not reveal the formation of higher molecular weight species, DLS analyses at two storage conditions revealed increases in the hydrodynamic radii and polydispersity index of the reconstituted formulations, suggesting the onset of formation of smaller species in the formulations. CD spectroscopy did not reveal any differences in the secondary structure of the mAb for the two concentrations after lyophilization. In conclusion, the freezing step method impacted the extent of glycation in lyophilized samples and the hydrolyzed component of sucrose was critical for increasing glycation. Even though some level of glycation was observed in lyophilized samples, the native structure of the protein was not affected. Further, it was demonstrated that storage of both lyophilized cakes and reconstituted formulations at higher temperatures could increase the extent of glycation in monoclonal antibody formulations. [ABSTRACT FROM AUTHOR]

Consistent high-quality antibody yield is a key goal for cell culture bioprocessing. This endpoint is typically achieved in commercial settings through product and process engineering of bioreactor parameters during development. When the process is complex and not optimized, small changes in composition and control may yield a finished product of less desirable quality. Therefore, changes proposed to currently validated processes usually require justification and are reported to the US FDA for approval. Recently, design-of-experiments-based approaches have been explored to rapidly and efficiently achieve this goal of optimized yield with a better understanding of product and process variables that affect a product's critical quality attributes. Here, we present a laboratory-scale model culture where we apply a Plackett- Burman screening design to parallel cultures to study the main effects of 11 process variables. This exercise allowed us to determine the relative importance of these variables and identify the most important factors to be further optimized in order to control both desirable and undesirable glycan profiles. We found engineering changes relating to culture temperature and nonessential amino acid supplementation significantly impacted glycan profiles associated with fucosylation, β-galactosylation, and sialylation. All of these are important for monoclonal antibody product quality. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1919-1928, 2015 [ABSTRACT FROM AUTHOR]

The viral and transmissible spongiform encephalopathy (TSE) safety of therapeutics of biological origin (biologicals) is greatly influenced by the nature and degree of variability of the source material and by the mode of purification. Plasma-derived and recombinant DNA products currently have good viral safety records, but challenges remain. In general, large enveloped viruses are easier to remove from biologicals than small ‘naked’ viruses. Monoclonal antibodies and recombinant DNA biopharmaceuticals are derived from relatively homogeneous source materials and purified by multistep schemes that are robust and amenable to scientific analysis and engineering improvement. Viral clearance is more challenging for blood and cell products, as they are complex and labile. Source selection (e.g. country of origin, deferral for CJD risk factors) currently occupies the front line for ensuring that biologicals are free of TSE agents, but robust methods for their clearance from products are under development. [Copyright &y& Elsevier]

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral "spiking" studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies.

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett-Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow-up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163-170, 2017., (© 2016 American Institute of Chemical Engineers.)

Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real-time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration-related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile., (© 2015 American Institute of Chemical Engineers.)

The size, heterogeneity, and biological production process of protein therapeutics like monoclonal antibodies create unique challenges for their analysis and regulation compared with small molecules. Complete structural characterization of a molecule 1000-fold heavier than aspirin is no small feat. Biological post-translational modifications such as glycosylation further complicate their characterization and regulation. Even approved protein therapeutics are known to contain multiple structural variants in differing amounts. Structural modification occurs during production and storage as well as within patients after administration. Thus, the goals of manufacturers and regulators are to control and characterize this heterogeneity, not take on the impossible task of eliminating it. The aim of this review is to describe the structural heterogeneities known to occur with immunoglobulin G (IgG), note current detection and analytical strategies, establish their causes, and define their potential effects on the ultimate safety, purity, and potency of antibody therapeutics when known.