Cold denaturation of monoclonal antibodies

Lazar, Kristi L.; Patapoff, Thomas W.; Sharma, Vikas

doi:10.4161/mabs.2.1.10787

Cited by 49 publications

(45 citation statements)

References 57 publications

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“…Unfortunately, protein destabilization and/or aggregation can occur either during storage or during the process of freezing or thawing. Using chemical denaturation and low pH destabilization, a set of persuasive studies were conducted to determine the temperatures at which maximal thermodynamic stability of an mAb and at which cold denaturation occurs [ 23 ]. Using two mAbs that only differed in their complementarity determining regions and some flanking amino acids, the authors concluded that these questions must be answered empirically for each mAb.…”

Section: Discussionmentioning

confidence: 99%

Bench to Bedside: Stability Studies of GMP Produced Trastuzumab-TCMC in Support of a Clinical Trial

2015

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The first-in-human phase 1 clinical radioimmunotherapy (RIT) trial with 212Pb-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane-trastuzumab (212Pb-TCMC-trastuzumab) was completed in October 2014 as a joint effort at the University of Alabama (UAB) and the University of California San Diego Moores Cancer Center. The preliminary reports indicate that after five dose-levels of intraperitoneally administered 212Pb-TCMC-trastuzumab, patients with carcinomatosis experienced minimal agent-related toxicity. This report presents the data accumulated to date on the stability of the clinical grade, produced according to current good manufacturing practices (cGMP), TCMC-trastuzumab conducted in support of that clinical trial. Of the eleven tests performed with the cGMP TCMC-trastuzumab all but one remained within specifications throughout the 5 year testing period. The protein concentration varied by 0.01 mg/mL at 48 months. Two other assays, ion-exchange high performance liquid chromatography (IEX-HPLC) and a competitive radioimmunoassay (RIA) indicated that the cGMP TCMC-trastuzumab integrity may be changing, although the change thus far is within specifications. Subsequent stability testing will confirm if a trend has truly developed. The cGMP TCMC-trastuzumab was also evaluated for tolerance to higher temperatures and the potential of storage at −80 °C. The immunoconjugate proved stable when subjected to the lower temperatures and to multiple freeze-thaw cycles. The size exclusion (SE) HPLC analysis of the 203Pb-TCMC-trastuzumab was the only indicator that cGMP TCMC-trastuzumab may be sensitive to storage at 37 °C for 3 months.

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Section: Discussionmentioning

confidence: 99%

Bench to Bedside: Stability Studies of GMP Produced Trastuzumab-TCMC in Support of a Clinical Trial

2015

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“…It is known that IgG shows more complex unfolding by equilibrium monitored methods 72 . To determine a kinetic isothermal denaturation curve for the multi-domain IgG molecule, a set of hydrated amine coupling human IgG coupled fully functional biosensor tips (supplied by forte BIO) were simultaneously exposed to increasing concentrations of either urea or GnHCl during a 5-min denaturant pulse.…”

Section: Resultsmentioning

confidence: 99%

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins

et al. 2016

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Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules/solution conditions that can stabilize or destabilize thermally stable proteins, multi-domain proteins, oligomeric proteins, and most importantly, aggregation prone metastable proteins.

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“…were also investigated but showed worse fit quality than the three-state model after an f-test comparison. The three-state model was also used by other groups to evaluate isothermal chemical denaturation data with mAbs 11,21 . Since all samples were evaluated with the three-state model, two Cm values were determined -Cm1 for the transition taking place at lower denaturant concentration and Cm2 for the other transition.…”

Section: Isothermal Chemical Denaturation (Icd)mentioning

confidence: 99%

A New Approach to Study the Physical Stability of Monoclonal Antibody Formulations—Dilution From a Denaturant

Svilenov

Gentiluomo

Frieß

et al. 2018

Journal of Pharmaceutical Sciences

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The early-stage assessment of the physical stability of new monoclonal antibodies in different formulations is often based on high-throughput techniques that suffer from various drawbacks. Accordingly, new approaches that facilitate the protein formulation development can be of high value to the industry. In this study, a dynamic light scattering plate reader is used to measure the aggregation (by means of the increase in the hydrodynamic radius [R]) of monoclonal antibody samples that were subject to incubation and subsequent dilution from different concentrations of a denaturing agent, that is, guanidine hydrochloride. The increase in the R of the protein samples is dependent not only on the denaturant concentration used but also on the buffer in which the incubation/dilution was performed. We also compare the aggregation after dilution from a denaturant with other high-throughput stability-indicating methods and find good agreement between the techniques. The proposed approach to probe the physical stability of monoclonal antibodies in different formulation conditions offers a unique combination of features-it is isothermal, probes both the resistance to denaturant-induced unfolding and the colloidal protein stability, it is entirely label-free, does not rely on complex data evaluation, and requires very short instrument measurement time on standard equipment.

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Cold denaturation of monoclonal antibodies

Cited by 49 publications

References 57 publications

Bench to Bedside: Stability Studies of GMP Produced Trastuzumab-TCMC in Support of a Clinical Trial

Bench to Bedside: Stability Studies of GMP Produced Trastuzumab-TCMC in Support of a Clinical Trial

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins

A New Approach to Study the Physical Stability of Monoclonal Antibody Formulations—Dilution From a Denaturant

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