A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

Understanding protein stability is central to combatting protein aggregation diseases and developing new protein therapeutics. At the high concentrations often present in biological systems, purified proteins can exhibit undesirable high solution viscosities and poor solubility mediated by short-range electrostatic and hydrophobic protein-protein interactions. The interplay between protein amino acid sequence, protein structure and solvent conditions to minimize protein-protein interactions is key to design of well-behaved pharmaceutical proteins. However, theoretical approaches have yet to yield a general framework to address these problems. Here, we analyzed the high concentration behavior of super-folder GFP (sfGFP) and two super-charged sfGFP variants engineered to have formal charges of −18 or +15. Under low cosolute conditions, sfGFP and the −18 variant formed a gel or phase separated at ~10 mg/ml. Under conditions that screen surface charges, including formulations with high histidine or high NaCl concentrations, all three variants attained concentrations up to 250 mg/ml with moderate viscosities. Moreover, all three variants exhibited very similar viscosity-concentration profiles over this range. This effect was not mimicked by high sugar concentrations that exert excluded volume effects without shielding charge. Collectively, these data demonstrate that charge shielding not only neutralizes long-range electrostatic interactions but also, surprisingly, short-range electrostatic effects due to surface charge anisotropy. This work shows that supercharged sfGFP behavior under high ionic strength is largely determined by particle geometry, a conclusion that is supported by colloid models and may be applicable to pharmaceutically-relevant proteins.

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“…Thus, the main contribution of trehalose appears to be its well-known role as a protectant during lyophilization. 51–52 …”

Section: Resultsmentioning

confidence: 99%

Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration

et al. 2017

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“…In addition to the detergent, some proteins require the addition of glycerol to stabilize the protein during the immobilization process ( Fig. 2 ) 35 . Although this has to be empirically determined, we have found that the addition of 10% (vol/vol) glycerol to the solublization buffer enhances the stability of the target protein 35 .…”

Section: Methodsmentioning

confidence: 99%

“…2 ) 35 . Although this has to be empirically determined, we have found that the addition of 10% (vol/vol) glycerol to the solublization buffer enhances the stability of the target protein 35 . Finally, in some cases, the protein stability is highly dependent on the lipid environment.…”

Section: Methodsmentioning

confidence: 99%

The preparation and development of cellular membrane affinity chromatography columns

Moaddel

Wainer

2009

Nat Protoc

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Cellular membrane affinity chromatography is a technique that is based on the immobilization of a target trans-membrane protein onto a stationary phase. The target protein is isolated by homogenization and solubilization of a source (e.g., cell line) followed by immobilization on either the immobilized artificial membrane-phosphatidyl choline (IAM-PC) stationary phase or the surface of an open tubular capillary during a dialysis step. The procedure typically takes 3–4 d for the IAM-PC stationary phase, whereas the open-tubular method takes an extra week for the preparation of the capillary. The resulting columns can then be used to characterize binding sites on the target protein through frontal chromatographic and/or nonlinear chromatographic studies using a wide variety of ligands including small molecules and polypeptides. The columns have been used in drug discovery as well as in the screening of tobacco smoke condensates.

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“…The impact of lyoprotectants on the stability of trastuzumab was reported [27]. The stability of ready-to use trastuzumab infusion solutions was studied by size-exclusion HPLC, UV-Vis spectrometry and SDS-PAGE [28].…”

Section: Introductionmentioning

confidence: 99%

Quality Control and Stability Studies with the Monoclonal Antibody, Trastuzumab: Application of 1D- vs. 2D-Gel Electrophoresis

Nebija¹,

Noe

Urban

et al. 2014

IJMS

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Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.

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A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

Cited by 261 publications

References 28 publications

Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration

Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration

The preparation and development of cellular membrane affinity chromatography columns

Quality Control and Stability Studies with the Monoclonal Antibody, Trastuzumab: Application of 1D- vs. 2D-Gel Electrophoresis

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