Efficient and economical purification platform for production of therapeutic nanobodies.

Single-domain antibodies, known as nanobodies, have arisen as an exciting class of biomolecules with applications ranging from diagnostics to numerous therapies. Due to their small size, enhanced stability, and reduced production complexity compared to traditional monoclonal antibodies, nanobodies present an economically attractive option with considerable commercial promise. In this paper, we describe a robust, efficient, and cost-effective downstream purification platform for production of nanobody-based biotherapeutics produced in Escherichia coli (E.

Utilizing liquid chromatography-mass spectrometry to map targeting of snake venom components by antivenom.

Preclinical efficacy testing is an essential aspect of evaluating quality of antivenoms (AVs). Recent years have witnessed a surge in development of in vitro methods to replace or reduce reliance on the standard mouse lethality assay. In this study, we propose a novel, reversed phase liquid chromatography-mass spectrometry (RPLC-MS)-based platform for monitoring AV activity on venom components under the WHO recommended in solution AV testing conditions.

Near-infrared spectroscopy coupled with convolutional neural network as a checkpoint tool for cell culture bioprocess media characterization.

As per the quality by design (QbD) paradigm, manufacturers are expected to identify critical raw materials that can contribute to variability in process performance and product quality. Further, manufacturers should be able to characterize and monitor the quality of these critical raw materials. Cell culture medium is universally accepted to be one such critical raw material for monoclonal antibody production.

Profiling Hinge Plasticity in Intact Monoclonal Antibodies for Antigen Recognition.

Monoclonal antibodies (mAbs) are multidomain glycosylated proteins that mediate antigen binding, among other protein-protein interactions, which makes them successful therapeutics. However, in many cases, adverse physicochemical properties can affect their antigen-binding abilities, compromising therapeutic potential. Hence, structural characterization of these biotherapeutics is strongly desired to predict and possibly redesign better variants.

Impact of Initial Aggregate Level on Aggregation Potential of Monoclonal Antibodies in Different Buffer Systems.

Purpose: This study investigates the stability and kinetics of degradation when monoclonal antibodies (mAbs) process intermediates are stored in commonly used Protein A elution buffers, including citrate, acetate, and glycine, at varying pre-existing aggregates levels (low: 1-5%, moderate: 5-15% and high: 15-25%) at 4°C and 30°C to simulate standard and worst-case conditions.

Methodology: mAb samples were subjected to thermal stress to achieve different levels of initial aggregates. The pre-aggregated samples were then incubated in different buffers at 4°C and 30°C to assess aggregation rates and stability.

Separation of Fab therapeutic charge variants by ion-exchange chromatography using iterative mathematical and artificial neutral network modeling approaches.

Linear pH, salt, or dual pH-salt gradient elution is the most common ion-exchange chromatography method for monoclonal and Fab antibody purification, but maintaining precise gradients during biomanufacturing is challenging. In the present study, using chromatographic data of linear salt gradient elution of Fab therapeutic performed at different conditions of pH and linear gradient lengths, a step gradient elution has been developed using iterative mathematical and artificial neutral networks modeling approaches. The proposed approaches utilizes classical Yamamoto method and Mollerup's thermodynamic approach offering satisfactory prediction of distribution coefficient of protein species (main Fab and two A1 and A2 acidic variants) as a function of salt concentration based on the stoichiometric displacement model for different fixed pH conditions.

Removal of Excipients from Drug Product may Impact Antibody Characterization of Monoclonal Antibodies.

Extensive analytical and functional characterization of a biotherapeutic product is a regulatory requirement, more so for biosimilar products where approval is contingent on the manufacturer's ability to demonstrate comparability of their product to the corresponding reference product. Typical biotherapeutic formulations contain multiple excipients that are meant to stabilize the product and these can impact certain analytical and functional techniques that are typically used in the above-mentioned characterization and comparability exercises. In this study, we elucidate this interference using Trastuzumab (Tmab) reference product, Herclon, and its commercially available biosimilars, Herzuma and Vivitra, as an example.

Disassembly mediated multimodal chromatography based purification of HPV-VLPs produced in Pichia pastoris.

Human Papillomavirus Virus-Like Particles (HPV-VLPs) are a highly effective vaccine to prevent cervical cancer. Current production and purification processes for HPV-VLPs suffer from poor yield and suboptimal process economics. The current study presents a purification strategy based multi-modal cation exchange chromatography (Capto™ MMC) for the purification of HPV-VLPs produced in Pichia pastoris.

Flocculation-based clarification for production of protein therapeutics in Pichia pastoris: Recombinant human serum albumin as a case study.

Pichia pastoris has been used as an expression system for multiple biotherapeutic products due to the unique advantages it offers with respect to cell density, protein titer, extracellular expression, and other such advantages. However, clarification of cell broth presents a significant challenge, primarily due to the high cell density (up to 50% W/V). Additionally, the abundance of host cell proteins complicates secondary clarification, impacting subsequent chromatographic, and filtration steps.

An Automated Tool for Glycosimilarity Assessment of mAb Therapeutic Biosimilars: Trastuzumab and Bevacizumab as Case Studies.

Background: With the expiration of patents for multiple biotherapeutics, biosimilars are gaining traction globally as cost-effective alternatives to the original products. Glycosylation, a critical quality attribute, makes glycosimilarity assessment pivotal for biosimilar development. Given the complexity of glycoanalytical profiles, assessing glycosimilarity is nontrivial.

Effect of formulation composition on trastuzumab stability.

For monoclonal antibody drug products as for other biologics, while the innovator drug products first becomes commercially available, they are often followed by one or more biosimilar products. These biosimilars often differ from the innovator product, as well as from each other, in their formulation composition. However, the impact of the formulation composition on the stability of the active pharmaceutical ingredient subjected to different 'stresses' is still not understood.

Impact of Various Forced Oxidative Stress Factors in Rapid Degradation of mAb: Trastuzumab as a Case Study.

Purpose: Therapeutic monoclonal antibodies (mAbs) are prone to degradation via aggregation and fragmentation. In this study, forced degradation of trastuzumab (TmAb) was explored in saline and in-vitro models having HO and exposed to UV light (case study 1) both bleomycin (BML) formulation and ferrous ions (Fe) (case study 2) and sodium hypochlorite (NaOCl) (case study 3).

Methods: Size exclusion chromatography, dynamic light scattering, spectroscopic analysis, and fluorescence microscope image processing was carried out for characterizing TmAb degradation.

N-Glycosylation modulators for targeted manipulation of glycosylation for monoclonal antibodies.

Monoclonal antibodies are extensively used as biotherapeutics for treatment of a variety of diseases. Glycosylation of therapeutic antibodies is considered a critical quality attribute as it influences the effector function, circulatory half-life, immunogenicity, and eventually efficacy and patient safety. During upstream process development, media components play a significant role in determining the glycosylation profile.

Higher concentration of trehalose dihydrate stabilizes recombinant IgG1 under forced stress conditions.

Stability of complex biotherapeutics like monoclonal antibodies is paramount for their safe and efficacious use. Excipients are inactive ingredients that are added to the purified product so as to offer it a stable environment. Trehalose dihydrate is a non-reducing sugar that is commonly used as a stabilizing agent in biotherapeutic formulations under liquid and frozen states.

Implementation of machine learning tool for continued process verification of process chromatography unit operation.

Recent advancements in technology, such as the emergence of artificial intelligence (AI) and machine learning (ML), have facilitated the progression of the biopharmaceutical industry toward the implementation of Industry 4.0. As per the guidelines set by the USFDA, process validation for biopharmaceutical production consists of three stages: process design, process qualification, and continuous process verification (CPV).

Challenges with effective removal of surfactants from monoclonal antibody formulations.

Buffer exchange is a critical step in biologics development, playing a pivotal role in removing contaminants, adjusting sample conditions, and facilitating compatibility studies. The efficiency of centrifugal concentrators for polysorbate removal was compared to a two-step approach involving a surfactant removal column followed by buffer exchange. Trastuzumab-pkrb from Herzuma® was used.

Development of a novel capture step for purification of antigen binding fragments (Fabs).

Antigen binding fragments (Fabs) are an emerging class of biotherapeutics, widely accepted as an alternative to the traditional monoclonal antibodies (mAbs). The small size of the Fabs offers better tissue penetrability and lack of Fc region, thereby resulting in reduced side effects. However, since Fab molecules lack Fc region, Protein A chromatography (the ubiquitous capture step in mAb platforms) cannot be employed.

MMIT-DDPM - Multilateral medical image translation with class and structure supervised diffusion-based model.

Unified translation of medical images from one-to-many distinct modalities is desirable in healthcare settings. A ubiquitous approach for bilateral medical scan translation is one-to-one mapping with GANs. However, its efficacy in encapsulating diversity in a pool of medical scans and performing one-to-many translation is questionable.

Elucidation of Mg induced size and charge heterogeneity in monoclonal antibody therapeutics.

Changes in charge variant profile are known to affect mAb stability and vice versa. This report elucidates the effects of magnesium metal (0.5 mM Mg) on trastuzumab (IgG1 antibody).

The economics of translating a biosimilar from lab to market in India.

This study aims to establish a cost basis for biologics manufacturers and policymakers by quantifying the price and time required to bring a biosimilar from the lab to market. For efficient implementation of a cost-based policy, especially for life-saving medicines like biosimilars, it is imperative to establish a benchmark for the cost involved in biosimilar development. In this holistic and multiple-case study, stage-wise cost estimates of biosimilar development were obtained for microbial and mammalian systems.

Offline Coupling of Hydrophobic Interaction Chromatography-Capillary Zone Electrophoresis for Monitoring Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies.

A holistic understanding of the charge heterogeneity in monoclonal antibodies (mAbs) is paramount for ensuring acceptable product quality. Hence, biotherapeutic manufacturers are expected to thoroughly characterize their products via advanced analytical techniques. Recently, two-dimensional liquid chromatography (2DLC) methods have gained popularity for resolving complex charged species.

Aggregation of therapeutic monoclonal antibodies due to thermal and air/liquid interfacial agitation stress: Occurrence, stability assessment strategies, aggregation mechanism, influencing factors, and ways to enhance stability.

Therapeutic proteins, such as monoclonal antibodies (mAbs) are known to undergo stability related issues during various stages of product life cycle resulting in the formation of aggregates and fragments. Aggregates of mAb might result in reduced therapeutic activity and could cause various adverse immunogenic responses. Sample containing mAb undergo aggregation due to various types of stress factors, and there is always a continuous interest among researchers and manufacturers to determine the effect of different factors on the stability of mAb.

Weakly supervised large-scale pancreatic cancer detection using multi-instance learning.

Introduction: Early detection of pancreatic cancer continues to be a challenge due to the difficulty in accurately identifying specific signs or symptoms that might correlate with the onset of pancreatic cancer. Unlike breast or colon or prostate cancer where screening tests are often useful in identifying cancerous development, there are no tests to diagnose pancreatic cancers. As a result, most pancreatic cancers are diagnosed at an advanced stage, where treatment options, whether systemic therapy, radiation, or surgical interventions, offer limited efficacy.

Recent advancements in soluble expression of recombinant antibody fragments in microbial host systems.

Recombinant fabs dominate the pharmaceutical pipelines today with microbial host systems continuing to be a major contributor toward their production. is a versatile host for recombinant protein expression due to its simplicity, affordability, and ability to be cultivated at high cell density. It is particularly suitable for non-glycosylated proteins and small proteins.

Role of Charge Heterogeneity on Physical Stability of Monoclonal Antibody Biotherapeutic Products.

Purpose: Chemical modifications in monoclonal antibodies can change hydrophobicity, charge heterogeneity as well as conformation, which eventually can impact their physical stability. In this study, the effect of the individual charge variants on physical stability and aggregation propensity in two different buffer conditions used during downstream purification was investigated.

Methods: The charge variants were separated using semi-preparative cation exchange chromatography and buffer exchanged in the two buffers with pH 6.