Purpose
To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates.

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Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates

In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al, Pharm Res 6:903-918, 1989), which has been widely referenced ever since At the time, recombinant protein therapy was still in its infancy This review summarizes the advances that have been made since then regarding protein stabilization and formulation In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability

Stability of Protein Pharmaceuticals: An Update

Protein aggregation--pathways and influencing factors.

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(10)00031-4.pdf

Recent Advances in Large-Scale Production of Monoclonal Antibodies and Related Proteins

Protein–excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development

There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin-G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates of between 20,000 and 250,000 s-1 for between 5 minutes and 30 ms using a parallel-plate and capillary rheometer respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s-1 and 0.06 pN respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel-plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat-induced aggregates of a mAb. We calculate that that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air-water interface or the 20 to 150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases air-bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production.

Response of a concentrated monoclonal antibody formulation to high shear.

Tungsten-induced protein aggregation: Solution behavior

Field flow fractionation (FFF) is a technique that holds great promise for the analysis and characterization of protein aggregates and particles, due to its wide dynamic range and matrix-free separation mechanism. FFF can be routinely used to achieve good monomer-oligomer separation and quantification for a variety of protein types, and is a reasonable choice for an orthogonal method for size exclusion chromatography and analytical ultracentrifugation. Quantifying sub-micrometer particles in protein therapeutics is a potential of the FFF technique that is yet to be realized, due to the lack of detection with sufficient sensitivity. In this article the effect of several important parameters on the optimization of FFF analyses are explored, and the strengths, weaknesses, and potential new applications of the technique are discussed.

Separation and characterization of protein aggregates and particles by field flow fractionation.

Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution

The development lifecycle for pharmaceutical proteins begins with target identification and demonstration of the biological relevance of a particular protein or protein property, continues to identification of which lead product candidate and cell line to advance, then proceeds through process and formulation development and characterization, clinical trials and commercialization. The launch of a product represents the beginning of a different kind of product support, which includes lot release, exploration of different delivery devices, comparability, and support for investigations. The past several decades have seen demonstration and documentation of the utility of asymmetrical flow field-flow fractionation (AF4) in biotechnology applicable to each of these protein drug development phases, but as yet, with limited industrial or routine implementation. This chapter seeks to provide a survey of such applications and potential opportunities for inspiration and exploitation of the distinct characteristics of AF4 throughout the long, winding and multifaceted drug development process.

Joey Pollastrini

Papers

Response of a concentrated monoclonal antibody formulation to high shear.

Tungsten-induced protein aggregation: Solution behavior

Separation and characterization of protein aggregates and particles by field flow fractionation.

Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution

Field-Flow Fractionation in Therapeutic Protein Development