Tapan Das

Identification (ID) testing is a regulatory requirement for biopharmaceutical manufacturing, requiring robust, GMP-qualified assays that can distinguish the therapeutic from any other in the facility. Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical tool used to identify and characterize biologics.
The proposed strategy is shown to be applicable for over 40 diverse model biologics including monoclonal antibodies (mAbs), biobetters such as antibody… Show more

Identification (ID) testing is a regulatory requirement for biopharmaceutical manufacturing, requiring robust, GMP-qualified assays that can distinguish the therapeutic from any other in the facility. Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical tool used to identify and characterize biologics.
The proposed strategy is shown to be applicable for over 40 diverse model biologics including monoclonal antibodies (mAbs), biobetters such as antibody prodrugs/afucosylated mAbs, fusion proteins, multi-specific antibodies, Fabs, and large peptides, all with excellent mass accuracy (error typically < 20 ppm) and precision. It requires a single-step sample preparation and a single click to run and process the data upon method setup Show less

Protein stability against aggregation is a major quality concern for the production of safe and effective biopharmaceuticals. Amongst the different drivers of protein aggregation, increasing evidence indicates that interactions between proteins and interfaces represent a major risk factor for the formation of protein aggregates in aqueous solutions. Potentially harmful surfaces relevant to biologics manufacturing and storage include air-water and silicone oil-water interfaces as well as… Show more

Protein stability against aggregation is a major quality concern for the production of safe and effective biopharmaceuticals. Amongst the different drivers of protein aggregation, increasing evidence indicates that interactions between proteins and interfaces represent a major risk factor for the formation of protein aggregates in aqueous solutions. Potentially harmful surfaces relevant to biologics manufacturing and storage include air-water and silicone oil-water interfaces as well as materials from different processing units, storage containers, and delivery devices. The impact of some of these surfaces, for instance originating from impurities, can be difficult to predict and control. Moreover, aggregate formation may additionally be complicated by the simultaneous presence of interfacial, hydrodynamic and mechanical stresses, whose contributions may be difficult to deconvolute. As a consequence, it remains difficult to identify the key chemical and physical determinants and define appropriate analytical methods to monitor and predict protein instability at these interfaces. In this review, we first discuss the main mechanisms of surface-induced protein aggregation. We then review the types of contact materials identified as potentially harmful or detected as potential triggers of proteinaceous particle formation in formulations and discuss proposed mitigation strategies. Finally, we present current methods to probe surface-induced instabilities, which represent a starting point towards assays that can be implemented in early-stage screening and formulation development of biologics. Show less

In spite of extensive research, protein aggregation still remains one of the most difficult phenomena to be understood in the field of biologics research and development. Protein aggregation is a complex process which results in the formation of a variety of supramolecular protein structures. Nucleation is the core step that initiates the cascade of molecular events leading to the formation of protein aggregates. Understanding and characterizing nucleation is therefore crucial to avoid… Show more

In spite of extensive research, protein aggregation still remains one of the most difficult phenomena to be understood in the field of biologics research and development. Protein aggregation is a complex process which results in the formation of a variety of supramolecular protein structures. Nucleation is the core step that initiates the cascade of molecular events leading to the formation of protein aggregates. Understanding and characterizing nucleation is therefore crucial to avoid undesired protein aggregation. Here we review the state of the art on protein aggregation in biotherapeutics, primarily focusing on the nucleation events, stimulating discussions about key open questions, and clarifying the peculiarities of aggregation process relative to other protein phase separation processes, such as crystallization. We summarize recent progress in the identification of the sources of protein aggregation and in the development of analytical tools to characterize this process. Moreover, we discuss significant gaps in the analysis and understanding of nucleation in non-native aggregation of biologics. Show less

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the… Show more

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs. Show less

Monoclonal antibodies are heterogeneous in nature and may contain numerous variants with differences in size, charge, and hydrophobicity, which may impact clinical efficacy, immunogenicity, and safety. Characterization of antibody variants is necessary to build structure-function correlation and establish a proper control strategy. Isolation and enrichment of variants by conventional chromatographic peak fractionation is labor-intensive and time-consuming. The instability of fractions during… Show more

Monoclonal antibodies are heterogeneous in nature and may contain numerous variants with differences in size, charge, and hydrophobicity, which may impact clinical efficacy, immunogenicity, and safety. Characterization of antibody variants is necessary to build structure-function correlation and establish a proper control strategy. Isolation and enrichment of variants by conventional chromatographic peak fractionation is labor-intensive and time-consuming. The instability of fractions during isolation and subsequent characterization may also be a concern. Hence, it is desirable to analyze antibody variants in an online and real-time manner. Here we demonstrate a 2D-LC methodology - multiple heart-cutting IEC-SEC- as an investigational tool to facilitate a charge variant characterization study. Both IEC modes - anion exchange (AEX) and cation exchange (CEX) chromatography are discussed. Using this approach, direct bridging of size and charge variants of an antibody molecule was achieved without offline peak fractionation. It was observed that antibody aggregates elute late on both the AEX and CEX columns, presumably due to secondary hydrophobic interactions. Additionally, we overcame the solvent mismatch issue and developed a 2D SEC-IEC method to confirm the bridging results. This is the first reported SEC-IEC 2D-LC application for the characterization of antibody size and charge variants. Show less

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings… Show more

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings together expertise and knowledge from biopharmaceutical scientists and discusses the various manufacturing process steps that could adversely impact the quality of drug substance (DS). The major process steps discussed here are commonly used in mAb production using mammalian cells. These include production cell culture, harvest, antibody capture by protein A, virus inactivation, polishing by ion-exchange chromatography, virus filtration, ultrafiltration-diafiltration, compounding followed by release testing, transportation and storage of final DS. Several of these process steps are relevant to protein DS production in general. The authors attempt to critically assess the level of risk in each of the DS processing steps, discuss strategies to control or mitigate protein modification in these steps, and recommend mitigation approaches including guidance on development studies that mimic the stress induced by the unit operations. Show less

somerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and… Show more

somerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and consequently establishing a robust control strategy. As a practical solution to this problem, a comprehensive analytical tool kit has been developed, which provides a solution to fully characterize and accurately quantify the Asp-related product variants. The toolkit offers a combination of 2 steps, an ion-exchange chromatography method to separate and enrich the isomerized variants in the folded structure for structure-function evaluation and a novel focused peptide mapping method to quantify the individual complementarity-determining region isomerization components including the unmodified Asp, succinimide, and isoaspartate. This novel procedure allowed an accurate quantification of each Asp-related variant and a comprehensive assessment of the functional impact of Asp isomerization, which ultimately helped to establish an appropriate control strategy for this critical quality attribute. Show less

Monoclonal antibodies are attractive therapeutic agents because of their impressive biological activities and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of chemical modifications, and the impact of such modifications on antibody physical stability and aggregation remains understudied. Here, we report a systematic analysis of the impact of methionine oxidation, tryptophan oxidation, and asparagine deamidation on antibody conformational and… Show more

Monoclonal antibodies are attractive therapeutic agents because of their impressive biological activities and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of chemical modifications, and the impact of such modifications on antibody physical stability and aggregation remains understudied. Here, we report a systematic analysis of the impact of methionine oxidation, tryptophan oxidation, and asparagine deamidation on antibody conformational and colloidal stability, hydrophobicity, solubility, and aggregation. Interestingly, we find little correlation between the impact of these chemical modifications on antibody conformational stability and aggregation. Methionine oxidation leads to significant reductions in antibody conformational stability while having little impact on antibody aggregation except at extreme conditions (low pH and elevated temperature). Conversely, tryptophan oxidation and asparagine deamidation have little impact on antibody conformational stability while promoting aggregation at a wide range of solution conditions, and the aggregation mechanisms appear linked to unique types of reducible and nonreducible covalent crosslinks and, in some cases, to increased levels of attractive colloidal interactions. These findings highlight that even related types of chemical modifications can lead to dissimilar antibody aggregation mechanisms, and evaluating these findings for additional antibodies will be important for improving the systematic generation of antibodies with high chemical and physical stability. Show less

The higher order structure (HOS) of proteins plays a critical role in the efficacy and stability of biological drugs. Perturbation of the regional structure of proteins can affect biological activity and cause instability. Characterization of HOS has become an integral part of biological drug development and is expected from regulatory agencies. The commonly used techniques for HOS characterization, such as circular dichroism, Fourier-transform infrared, differential scanning calorimetry… Show more

The higher order structure (HOS) of proteins plays a critical role in the efficacy and stability of biological drugs. Perturbation of the regional structure of proteins can affect biological activity and cause instability. Characterization of HOS has become an integral part of biological drug development and is expected from regulatory agencies. The commonly used techniques for HOS characterization, such as circular dichroism, Fourier-transform infrared, differential scanning calorimetry, intrinsic fluorescence, and hydrogen–deuterium exchange mass spectrometry, have their limitations ranging from lack of sensitivity and specificity to the need of high-level expertise and poor access to instrumentation due to high cost. In this study, we demonstrated a novel controlled proteolysis-based LC-QDa method for the detection of HOS change. By digesting proteins directly without denaturation and reduction, the HOS information can be revealed through the digested peptides. After optimizing the digestion conditions and the detection procedures, we identified 13 signature peptides that can monitor various antibody domains for any HOS changes caused by external stress. By comparing the peptide peak areas between unknown samples and a native control sample, any regional structural changes in unknown samples can be detected. The method was subsequently applied to a wide range of forced degradation samples to demonstrate higher sensitivity compared to the near-UV CD method that is frequently used for monitoring tertiary structural changes. By further reducing the number of signature peptides to five and optimizing liquid chromatography gradient duration, a streamlined, high-throughput, and controlled proteolysis method was successfully established. This method can be used to support process and formulation development as well as potentially for stability testing. Show less

Biophysical Methods for Biotherapeutics: Discovery and Development Applications
Tapan K. Das (Editor)
ISBN: 978-0-470-93843-0
376 pages
April 2014

DESCRIPTION:
With a focus on practical applications of biophysical techniques, this book links fundamental biophysics to the process of biopharmaceutical development.

• Helps formulation and analytical scientists in pharma and biotech better understand and use biophysical methods
• Chapters organized according to the… Show more

Biophysical Methods for Biotherapeutics: Discovery and Development Applications
Tapan K. Das (Editor)
ISBN: 978-0-470-93843-0
376 pages
April 2014

DESCRIPTION:
With a focus on practical applications of biophysical techniques, this book links fundamental biophysics to the process of biopharmaceutical development.

• Helps formulation and analytical scientists in pharma and biotech better understand and use biophysical methods
• Chapters organized according to the sequential nature of the drug development process
• Helps formulation, analytical, and bioanalytical scientists in pharma and biotech better understand and use strengths and limitations of biophysical methods
• Explains how to use biophysical methods, the information obtained, and what needs to be presented in a regulatory filing, assess impact on quality and immunogenicity
• With a focus on practical applications of biophysical techniques, this book links fundamental biophysics to the process of biopharmaceutical development. Show less

Formation of aggregates and particulates in biopharmaceutical formulation continues to be one of the major quality concerns in biotherapeutics development. The presence of large quantities of aggregates is believed to be one of the causes of unwanted immunogenic responses. Protein particulates can form in a wide range of sizes and shapes. Therefore, a comprehensive characterization of particulates in biologics formulation continues to be challenging. The quantity of small size aggregates (e.g.,… Show more

Formation of aggregates and particulates in biopharmaceutical formulation continues to be one of the major quality concerns in biotherapeutics development. The presence of large quantities of aggregates is believed to be one of the causes of unwanted immunogenic responses. Protein particulates can form in a wide range of sizes and shapes. Therefore, a comprehensive characterization of particulates in biologics formulation continues to be challenging. The quantity of small size aggregates (e.g., dimer) in a stable biologics formulation is well controlled using precision analytical techniques (e.g., high-performance liquid chromatography). Particulate in clinical and commercial formulations is monitored using visual inspection and subvisible particulate counting assays. While visual inspection (by human eye or automated systems) is intended to detect particulates (intrinsic and extrinsic) of ~100 μm or larger, the subvisible counting methods cover smaller size ranges down to 10 μm. It is well recognized that research of particulates in the submicron (<1 μm) and low-micron (1-10 μm) ranges may provide important clues to understand the mechanism of particulate formation. The recent years have seen a significant increase in the development of newer technologies for more comprehensive characterization of particulates. This is attributed to increased awareness in this field of research over the past 5 years, stimulated by scholarly articles, commentaries, and robust discussions in various forums. This article provides an overview of emerging detection technologies that provide complementary characterization data encompassing a wider size range of particulates. It also discusses their advantages and limitations in the context of applications in biotherapeutics development. Show less