Batch Consistency Research Articles

Degradomics-Based Analysis of Tetanus Toxoids as a Quality Control Assay.

Currently, batch release of toxoid vaccines, such as diphtheria and tetanus toxoid, requires animal tests to confirm safety and immunogenicity. Efforts are being made to replace these tests with in vitro assays in a consistency approach. Limitations of current in vitro assays include the need for reference antigens and most are only applicable to drug substance, not to the aluminum adjuvant-containing and often multivalent drug product. To overcome these issues, a new assay was developed based on mimicking the proteolytic degradation processes in antigen-presenting cells with recombinant cathepsin S, followed by absolute quantification of the formed peptides by liquid chromatography-mass spectrometry. Temperature-exposed tetanus toxoids from several manufacturers were used as aberrant samples and could easily be distinguished from the untreated controls by using the newly developed degradomics assay. Consistency of various batches of a single manufacturer could also be determined. Moreover, the assay was shown to be applicable to Al(OH)3 and AlPO4-adsorbed tetanus toxoids. Overall, the assay shows potential for use in both stability studies and as an alternative for in vivo potency studies by showing batch-to-batch consistency of bulk toxoids as well as for aluminum-containing vaccines.

The Importance of Evaluating the Lot-to-Lot Batch Consistency of Commercial Multi-Walled Carbon Nanotube Products.

The biological response of multi-walled carbon nanotubes (MWNTs) is related to their physicochemical properties and a thorough MWNT characterization should accompany an assessment of their biological activity, including their potential toxicity. Beyond characterizing the physicochemical properties of MWNTs from different sources or manufacturers, it is also important to characterize different production lots of the same MWNT product from the same vendor (i.e., lot-to-lot batch consistency). Herein, we present a comprehensive physicochemical characterization of two lots of commercial pristine MWNTs (pMWNTs) and carboxylated MWNTs (cMWNTs) used to study the response of mammalian macrophages to MWNTs. There were many similarities between the physicochemical properties of the two lots of cMWNTs and neither significantly diminished the 24-h proliferation of RAW 264.7 macrophages up to the highest concentration tested (200 μg cMWNTs/mL). Conversely, several physicochemical properties of the two lots of pMWNTs were different; notably, the newer lot of pMWNTs displayed less oxidative stability, a higher defect density, and a smaller amount of surface oxygen species relative to the original lot. Furthermore, a 72-h half maximal inhibitory concentration (IC-50) of ~90 µg pMWNTs/mL was determined for RAW 264.7 cells with the new lot of pMWNTs. These results demonstrate that subtle physicochemical differences can lead to significantly dissimilar cellular responses, and that production-lot consistency must be considered when assessing the toxicity of MWNTs.

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF.

Recombinant human IFNα2b (rhIFNα2b), as an important immune-related protein, has been widely used in clinic for decades. It is also at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Although with the same amino acid sequence, recombinant proteins are generally heterogeneous due to post-translational modification and chemical reactions during expression, purification, and long-term storage, which could have significant impact on the final product quality. So therapeutic rhIFNα2b must be closely monitored to ensure consistency, safety, and efficacy. In this study, we compared seven rhIFNα2b preparations from six manufacturers in China and one in America, as well as four batches of rhIFNα2b preparations from the same manufacturer, measuring IFNα2b variants and site-specific modifications using a developed LC/Q-TOF approach. Three main forms of N-terminus, cysteine, methionine, and acetylated cysteine were detected in five rhIFNα2b preparations produced in E. coli (1E~5E) and one in Pseudomonas (6P), but only the native form with N-terminal cysteine was found in rhIFNα2b preparation produced in Saccharomyces cerevisiae (7Y). Two samples with the lowest purity (4E and 6P), showed the highest level of acetylation at N-terminal cysteine and oxidation at methionine. The level of oxidation and deamidation varied not only between samples from different manufacturers but also between different batches of the same manufacturer. Although variable between samples from different manufacturers, the constitution of N-terminus and disulfide bonds was relatively stable between different batches, which may be a potential indicator for batch consistency. These findings provide a valid reference for the stability evaluation of the production process and final products.

Glycoconjugate vaccine batch consistency assessed by objective comparison of circular dichroism spectra

Circular dichroism (CD) spectra of biopharmaceutical protein, or protein conjugate, products contain information about their secondary and tertiary structures, which can answer to increasing regulatory interest in demonstrating consistent higher order structures of production batches. Widespread routine use of CD in a regulatory environment requires objective, statistically based, and validated methods to analyse and compare spectra against product specifications. Correlation approaches to compare spectra, developed and tested on monoclonal antibodies, are here used to assess the consistency of Hib PRP-CRM197 glycoconjugate immunogen batches, by analysis of historical data sets. Deconvolution of spectra into Gaussian peaks was used to model the spectrum and allow a more detailed description of spectral differences. Two groups of spectra [and hence samples] were distinguished. The analyses are discussed in the context of spectral comparison approaches, inter-laboratory studies, potential regulatory use and sources of uncertainty between spectra. Data analysis methods implemented here can also support stability and formulation studies.

An Imaging Toolkit for Physical Characterization of Long-Acting Pharmaceutical Implants

In pharmaceutical development alternative drug delivery modalities are being increasingly employed. One example is an implant, which achieves gradual drug release in patients over a period of many months or years. Due to the complexity of these long-acting formulations, advanced physical characterization methods are desirable as screening tools during protracted formulation development. Imaging methods are of particular interest due to their ability to interrogate the structure and composition of implants spatially across multiple length scales (macro, micro, nano). In this work, spatiochemical imaging is shown to interrogate many crucial drug product attributes of solid implants: overall implant structure, drug distribution, micro-domain size and orientation, agglomeration, porosity and defects, drug/excipient interface, dissolution process, and release mechanism. Imaging methods facilitate a detailed understanding of the process/structure correlation to inform on formulation selection, process parameter optimization, and batch consistency. Numerous case studies of implant applications with imaging are discussed. Methods utilized are X-ray computed tomography (XRCT), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS) imaging, and Raman microscopy. The imaging data is complemented with solid-state nuclear magnetic resonance (ssNMR). Altogether, these examples demonstrate that complementary imaging methods are highly effective for analyzing complex and novel pharmaceutical modalities such as solid implants.

Solubility of Pharmaceutical Ingredients in Natural Edible Oils.

Natural edible oils (NEOs) are common excipients for lipid-based formulations. Many of them are complex mixtures comprising hundreds of different triglycerides (TGs). One major challenge in developing lipid-based formulations is the variety in NEO compositions affecting the solubility of active pharmaceutical ingredients. In this work, solubilities of indomethacin (IND), ibuprofen (IBU), and fenofibrate (FFB) in soybean oil and in coconut oil were measured via differential scanning calorimetry, high-performance liquid chromatography, and Raman spectroscopy. Furthermore, this work proposes an approach that mimics NEOs using one key TG and models the API solubilities in these NEOs based on perturbed-chain statistical associating fluid theory (PC-SAFT). Key TGs were determined using the 1,2,3-random hypothesis, and PC-SAFT parameters were estimated via a group-contribution method. Using the proposed approach, the solubility of IBU and FFB was modeled in soybean oil and coconut oil. Furthermore, the solubilities of five more APIs (IND, cinnarizine, naproxen, griseofulvin, and felodipine) were modeled in soybean oil. All modeling results were found in very good agreement with the experimental data. The influence of different NEO kinds on API solubility was examined by comparing FFB and IBU solubilities in soybean oil and refined coconut oil. PC-SAFT was thus found to allow assessing the batch-to-batch consistency of NEO batches in silico.

Comparative study on dissolution behavior of Fufang Danshen Pian

To compare the quality of Fufang Danshen Pian, the dissolution behaviors of 7 index components in Fufang Danshen Pian in different batches and from different manufacturers in 4 kinds of dissolution mediums, and evaluate by multiple dissolution curves. The dissolution test was carried out by the paddle method, with pH 1.2 hydrochloride buffer, pH 4.0 acetate buffer, distilled water and pH 6.8 phosphate buffer as dissolution medium. The rotation speed was 75 r·min~(-1). The content of 7 index components at each sampling point was measured by HPLC. The accumulative dissolution rate was calculated, the cumulative dissolution curve was drawn, and the dissolution models were fitted. The dissolution behaviors and the dissolution curve fitting equations of Fufang Danshen Pian in different batches and from different manufacturers in different dissolution mediums were quite different. The dissolution of the same preparation from different manufactures was different in the same dissolution medium, and the dissolution of different index components from the same manufacturer was not synchronized in the same dissolution medium; the dissolution behaviors of Fufang Danshen Pian in different batches and from different manufacturers in the same dissolution medium were different and greatly affected by pH value, indicating differences in the quality of Fufang Danshen Pian in different batches and from different manufacturers. This study provides scientific basis for the improvement in preparation process and the evaluation of batch consistency of Fufang Danshen Pian.

Control over Polymorph Formation of Polydatin in Binary Solvent System and Structural Characterization

Polydatin is a natural product used for anti-oxidant, anti-inflammatory and anti-tumor purposes, and often added in medicine, nutraceutical, cosmetics, and dietary supplement. Polymorphism is a key feature of solid-state pharmaceutical products. Polymorphic modifications may exhibit different physical and chemical properties. Here we report two different polymorphs, and the amorphous form of Polydatin. Polymorphs were prepared in binary solvent system. The crystal structures of the two forms were revealed for the first time. The structure and 3D packing were determined with single crystal X-ray diffraction analysis. The batch consistency and stability were identified with Powder X-ray diffraction analysis. Various functional groups present in the polymorphs were analyzed with fourier transform infrared spectroscopic method. The thermal properties were investigated with DSC and TGA. HPLC-MS was used for the pharmacokinetic study. Results show that form B has the faster absorption, and can be maintained in animal bodies for a longer time than form A.

Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry

Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme.We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate–polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied.Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished.We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches.

Determination of size variants by CE-SDS for approved therapeutic antibodies: Key implications of subclasses and light chain specificities

In the present work, a generic non-reducing capillary electrophoresis sodium dodecyl sulphate (nrCE-SDS) method was tested for a wide range of 26 FDA and EMA approved monoclonal antibodies (mAbs) and 2 antibody drug conjugates (ADCs) as well as for the NISTmab, in a QC environment (e.g. testing quality requirements for batch manufacturing or batch release). This method allows obtaining rapidly and accurately the amount of size variants in drug products within about 40 min and may be used for batch release and consistency as well as for stability and shelf-life. First, the method repeatability was found to be excellent in terms of relative migration times and relative proportions of fragments (average RSD values of 0.3 and 0.2 %, on relative migration times and relative percentages of fragments, respectively), thanks to the addition of an internal standard. A panel of chimeric, humanized and human mAbs were tested, belonging to different subclasses (heavy chain gamma 1, 2, 2/4 and 4) and light chain types (κ or λ) and produced in different cell lines (CHO, NS0 and SP2/0). For all these biopharmaceutical products, the amount of H2L2 species was comprised between 90.9 % and 97.7 %, except for the two mAbs belonging to the IgG1λ subclass, namely avelumab and belimumab, which were prone to partial reduction during the sample preparation at 70 °C. Based on the CE-SDS results obtained for a diverse panel of therapeutic antibodies investigated in this study, and covering a wide range of structural and physico-chemical properties, a specification on the intact antibody content (H2L2) greater than 90 % can be achieved.

Evaluation of a multiple and global analytical indicator of batch consistency: traditional Chinese medicine injection as a case study.

This paper evaluates a multiple and global analytical indicator of batch consistency in traditional Chinese medicine injections (TCMIs) via a chemometrics tool, which is more comprehensive to appraise quality consistency of different batches of injections than the traditional method of fingerprint similarity. A commonly used TCMI, Salviae miltiorrhizae and ligustrazine hydrochloride injection (SLI), was employed as a model. With the aid of a chemometrics tool (principal component analysis, PCA), evaluation of multiple and global analytical indicators of batch consistency, which included saccharides, phenolic acids and inorganic salts (18 indicators in total), was carried out to appraise the quality consistency of 13 batches of injection provided by the Guizhou Baite Pharmaceutical Co., Ltd. (Guizhou, China). Compared with the traditional HPLC-UV fingerprint similarity evaluation, the method proposed in the paper can more comprehensively and correctly reflect the quality consistency of different batches of injections. In this paper, the multi-index evaluation result showed poor batch consistency, which was more consistent with the determination results, while the fingerprint similarity evaluation results still showed good batch consistency. The HPLC-UV fingerprint reflects only substances with UV absorption, but it is not able to reflect substances without UV absorption or weak UV absorption, which leads to inappropriate conclusions. Therefore, quality consistency of injections can be effectively appraised by evaluation of multiple and global analytical indicators, instead of HPLC-UV fingerprint only. For visualizing the batch consistency of the multiple and global analytical indicators, a heat map was used to represent the fluctuation. Furthermore, critical indicator identification was also applied to select several indicators that should be paid more attention during the process of quality control of injection. And the analysis result showed that Na+, fructose (Fru), glucose (Glc), manninotriose (Man), danshensu (DSS) and salvianolic acid B (SAB) are the indicators that should be given more attention when controlling the quality of injections, also called critical quality control indicators. The proposed method provides a reference for the quality control of TCMIs and has broad application potential.

Measurement of surface protein antigens, PorA and PorB, in Bexsero vaccine using quantitative mass spectrometry

Bexsero is a multivalent vaccine containing outer membrane vesicles (OMV) derived from Neisseria meningitidis group B strain NZ98/254 and three recombinant meningococcal proteins, Neisserial adhesin A, Heparin binding antigen and factor H binding protein. OMV production relies on the growth of large-scale cultures of N. meningitidis under controlled conditions. Changes to environmental factors, such as temperature, pH, nutrient availability and trace elements, can impact the growth rate of the meningococcus. Furthermore outer membrane expression levels vary in response to the environmental milieu, thus any changes in environmental conditions can result in changes in OMV protein content. This makes consistent production of OMVs challenging and the ability to measure the protein content of the final product is desirable to ensure product quality. The aim of this work was to develop a mass spectrometry (MS) method for measuring the porin proteins and to evaluate this approach for assessing the batch consistency of Bexsero vaccine. Using isotope dilution MS, we measured the PorA and PorB content in 75 lots of Bexsero vaccine. PorA ranged from 4.0 to 5.95 μg/dose with an average of 4.8 μg/dose. PorB ranged from 5.4 to 8.7 μg/dose with an average of 6.5 μg/dose. This is the first description of the quantitative characterisation of adjuvanted Bexsero vaccine drug product at the final stage of the production process, once the aluminium adjuvanted vaccine has been packaged into syringes, to assess manufacturing consistency. The significance of our findings to quality control in the future is discussed.

Hydrophobic interaction chromatography (HIC) method development and characterization of resolved drug-load variants in site-specifically conjugated pyrrolobenzodiazepine dimer-based antibody drug conjugates (PBD-ADCs)

Antibody drug conjugates (ADCs) are heterogeneous biopharmaceutical products that demand extensive characterization to ensure batch consistency, safety, and efficacy. Hydrophobic interaction chromatography (HIC) is the state-of-the-art analytical tool to monitor conjugation-related critical quality attributes (CQAs) e.g. drug-load distribution and Drug-to-Antibody Ratio (DAR). For the next generation site-specific PBD-ADCs (PBD: pyrrolobenzodiazepine dimer), denaturing RP-HPLC (reverse-phase high-performance chromatography) is the current method to determine average DAR. In this manuscript, we have utilized native HIC for the first time to understand conjugation related CQAs in PBD-ADCs. In terms of the method development, the type of stationary phase and salt, coupled with reduction of the reactive imine in the PBD drug-linker to an amine form in the sample preparation, have played a key role in achieving the best HIC resolution for the drug-load variants. The established HIC conditions resolved DAR 0, DAR 1, and two DAR 2 peaks for PBD-ADCs. Extended characterization of the DAR 2 peaks confirmed that they have retained characteristically distinct antibody Fc N-glycan distributions (Fc = Fragment crystallization region). Therefore, the results support that the HIC conditions established for PBD-ADCs is valuable in not only determining DAR values but also other important attributes including native drug-load distribution and unique DAR 2 conformations existed as a result of the N-glycan heterogeneity.

Luminescent Silica Nanosensors for Lifetime Based Imaging of Intracellular Oxygen with Millisecond Time Resolution.

Intracellular oxygen concentration was quantitatively imaged and rapidly traced with millisecond time resolution. We have demonstrated a new kind of oxygen nanosensors based on a ruthenium complex doped solid silica nanoparticles, which showed high oxygen sensing performance (I0/I100 = 3.29, t95 < 3 s) and ease of surface functionalization. Their sensing performance can be tuned by changing types of oxygen-sensitive probes and particle morphology. The nanosensors showed excellent control in both sensor size (from 30 to 200 nm), monodispersity, morphology, surface chemistry, and batch to batch consistency. Their uniform size distribution and good biocompatibility made them suitable for intracellular studies. Because the sensor surface can be easily functionalized with arbitrary units (such as transmembrane motifs, drugs, organelle-targeting groups, imaging reagent, and multiple sensor probes), these nanosensors provide a general platform to build easy-to-use tools for intracellular applications. The ease of surface functionalization was demonstrated by modifying the sensors outer surface with morpholinopropylamine and (3-carboxypropyl) triphenyl phosphonium, to actively target intracellular lysosomes and mitochondria of the tested cell lines (HeLa, MCF-7, and MCF-10A). Applying the mitochondria-targeting oxygen nanosensor together with our custom-built rapid phosphorescent lifetime imaging system, variations of intracellular oxygen have been quantitatively imaged and traced (in millisecond intervals) in real time and in situ.

Adsorption of mercury ions from synthetic aqueous solution using polydopamine decorated SWCNTs

Mercury is one of the most toxic and unnecessary elements for human body in a way that only slight amount of it can cause serious hygienic problems and environmental pollution. In the present study, magnetized single walled carbon nanotubes (SWCNTs) functionalized by polydopamine was employed for elimination of mercury heavy metal ions from aqueous media. In order to characterize the physical and chemical properties of the SWCNT and to confirm the functionalization steps, different analyses including TEM, SEM, VSM, EDS, FT-IR were utilized. In the present paper, the effects of experimental parameters such as adsorbent dosage, solution pH, metallic ion initial concentration, process thermodynamics and desorption mechanism on batch system were widely studied. In addition, mercury adsorption trend in continuous system was investigated by a fixed bed reactor. Comparison of adsorption process kinetics were done with models of Freundlich, Langmuir and Dubinin–Radushkevich in batch system and consistency with Yan and Thomas models in continuous system by Sigma plot and MATLAB software packs. Isotherm studies and adsorption kinetics revealed that mercury ions follow Freundlich adsorption isotherm and pseudo-second-order kinetic model. Moreover, thermodynamic studies showed that mercury ion adsorption is an exothermic and spontaneous reaction. Although Thomas model was found to predict more exact results for maximum mercury ion adsorption, both Thomas and Yan models had good fitness with fixed bed reactor data. Finally, adsorption process demonstrated that removal efficiency of the prepared adsorbent was so much higher than raw SWCNTs.

Validation of a Cell Proliferation Assay to Assess the Potency of a Dialyzable Leukocyte Extract Intended for Batch Release.

Transferon® is a blood product with immunomodulatory properties constituted by a complex mixture of peptides obtained from a human dialyzable leukocyte extract (DLE). Due to its complex nature, it is necessary to demonstrate batch consistency in its biological activity. Potency is the quantitative measure of biological activity and is also a quality attribute of drugs. Here we developed and validated a proliferation assay using Jurkat cells exposed to azathioprine, which is intended to determine the potency of Transferon® according to international guidelines for pharmaceuticals. The assay showed a linear response (2.5 to 40 µg/mL), coefficients of variation from 0.7 to 13.6% demonstrated that the method is precise, while r2 = 0.97 between the nominal and measured values obtained from dilutional linearity showed that the method is accurate. We also demonstrated that the cell proliferation response was specific for Transferon® and was not induced by its vehicle nor by other peptide complex mixtures (glatiramer acetate or hydrolyzed collagen). The bioassay validated here was used to assess the relative potency of eight released batches of Transferon® with respect to a reference standard, showing consistent results. The collective information from the validation and the assessment of several batches indicate that the bioassay is suitable for the release of Transferon®.

Simultaneous Quantification of Pharmacological Markers Quercetin and Berberine Using High-Performance Thin-Layer Chromatography (HPTLC) and High-Performance Liquid Chromatography (HPLC) from a Polyherbal Formulation Pushyanuga Churna.

Background: Ayurvedic medicines show great promise due to their holistic approach in the treatment of diseases. But proper standardization is necessary for their integration into mainstream medicine. One such well-known formulation is Pushyanuga Churna. As safety and efficacy of a multiherbal formulation is dependent on the authenticity of the ingredients used, chromatographic techniques play a significant role in the quality control of complex herbal medicines. Objective: In the current research, marker-based standardization of Pushyanuga Churna using validated HPTLC and HPLC methods have been established. Methods: Pushyanuga Churna was prepared in-house using authentic ingredients. Pharmacologically active biomarkers quercetin and berberine were simultaneously estimated using validated HPTLC and HPLC methods. Chromatographic fingerprints were developed for the formulation along with all the ingredients that can be used as tools to identify Pushyanuga Churna. Results: In-house formulation and Patanjali (marketed formulation) were observed to be rich in both markers. Quercetin was found to be more abundant than berberine in the ingredients. The deviation of marker content in marketed formulations can be attributed to variation in the ingredients used in the preparation. Conclusions: The routine use of such scientifically accepted methods will help in establishing the quality of the formulation and help in building confidence in traditional medicine. Highlights: The study highlights evaluation of biomarkers from the formulation and its ingredients as an important method to ensure the batch to batch consistency in quality of the formulation.

Simultaneous quantification and consistency analysis of 19 representative components in Longshengzhi Capsules by HPLC-QQQ/MS

Longshengzhi capsule consisting of 12 herbs is widely used in clinically treating cerebral ischemia during recovery period.In this study,in order to investigate the consistency of different batches of Longshengzhi capsules,a high performance liquid chromatography coupled to triple quadrupole mass spectrometry method(HPLC-QQQ/MS) was developed for the determination of 19 representative components in Longshengzhi Capsules within 9 min. Methodology validation indicated this method was simple,rapid,accurate,highly sensitive and reproducible,and it could be used for the content determination of components in Longshengzhi Capsules. The consistency analysis results showed that paeoniflorin and calycosin-7-glucoside in Longshengzhi Capsules had the highest content; RSD value of total content of 19 compounds was 5. 2% and the RSD value of main compounds such as astragaloside and calycosin-7-glucoside was all less than 15%,reflecting good consistency among different batches. This study has provided a scientific method and basis for the quality control and consistency evaluation of Longshengzhi Capsules.

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies.

Recombinant monoclonal antibodies (mAbs) intended for therapeutic usage are required to be thoroughly characterized, which has promoted an extensive effort towards the understanding of the structures and heterogeneity of this major class of molecules. Batch consistency and comparability are highly relevant to the successful pharmaceutical development of mAbs and related products. Small structural modifications that contribute to molecule variants (or proteoforms) differing in size, charge or hydrophobicity have been identified. These modifications may impact (or not) the stability, pharmacokinetics, and efficacy of mAbs. The presence of the same type of modifications as found in endogenous immunoglobulin G (IgG) can substantially lower the safety risks of mAbs. The knowledge of modifications is also critical to the ranking of critical quality attributes (CQAs) of the drug and define the Quality Target Product Profile (QTPP). This review provides a summary of the current understanding of post-translational and physico-chemical modifications identified in recombinant mAbs and endogenous IgGs at physiological conditions.

Measuring Particle Size Distribution by Asymmetric Flow Field Flow Fractionation: A Powerful Method for the Preclinical Characterization of Lipid-Based Nanoparticles.

Particle size distribution and stability are key attributes for the evaluation of the safety and efficacy profile of medical nanoparticles (Med-NPs). Measuring particle average size and particle size distribution is a challenging task which requires the combination of orthogonal high-resolution sizing techniques, especially in complex biological media. Unfortunately, despite its limitations, due to its accessibility, low cost, and easy handling, batch mode dynamic light scattering (DLS) is still very often used as the only approach to measure particle size distribution in the nanomedicine field. In this work the use of asymmetric flow field flow fractionation coupled to multiangle light scattering and dynamic light scattering detectors (AF4-MALS-DLS) was evaluated as an alternative to batch mode DLS to measure the physical properties of lipid-based nanoparticles. A robust standard operating procedure (SOPs) developed by the Nanomedicine Characterization Laboratory (EUNCL) was presented and tested to assess size stability, batch to batch consistency, and the behavior of the lipid-based nanoparticles in plasma. Orthogonal sizing techniques, such as transmission electron microscopy (TEM) and particle tracking analysis (PTA) measurements, were performed to support the results. While batch mode DLS could be applied as a fast and simple method to provide a preliminary insight into the integrity and polydispersity of samples, it was unsuitable to resolve small modifications of the particle size distribution. The introduction of nanoparticle sorting by field-flow fractionation coupled to online DLS and MALS allowed assessment of batch to batch variability and changes in the size of the lipid nanoparticles induced by the interaction with serum proteins, which are critical for quality control and regulatory aspects. In conclusion, if a robust SOP is followed, AF4-MALS-DLS is a powerful method for the preclinical characterization of lipid-based nanoparticles.