Predicting Long-term Stability of mAbs in Formulation Screenings using a Single, Fully Automated Platform

Published on December 20, 2016 at 10:27 AM

Introduction

Biologics is part of the rapidly growing group of drugs. New, predictive analytical methods are required to streamline the development process for an ever-increasing number of complex molecules, such as biosimilars, antibody-drug conjugates (ADCs) and mAbs. The long-term stability of a drug is a significant aspect of the development process (Geng et al, 2014; Seeliger et al, 2015).

The formation of protein particles is the most critical parameter, specifically at high concentrations. Thus, protein particles can act as nucleation seeds and might support protein aggregation.

As a result, particle formation could have undesired effects, rendering the drug ineffective or even harmful to patients in addition to reducing the amount of active native-like protein (Garidel, 2014; Roberts, 2014).

Assessing the relative thermal stability of biologics and measuring ΔG° of unfolding are becoming important tools during protein engineering and formulation development to estimate the aggregation propensity of biologics (Temel et al, 2016).

The chemical denaturation approach involves shifting of protein conformation equilibrium from a folded to a partially unfolded up to a fully unfolded state by raising the concentration of a chaotropic salt (guanidine hydrochloride (GuaHCl)).

The changes in the F350/F330 fluorescence ratio can be monitored to determine the unfolded protein fraction at each measured GuaHCl concentration. The resultant value can be used to calculate GuaHCl concentration dependence of unfolding (ΔG°) (Figure 1).

Importantly, the amount of fraction of -folded protein can also be very precisely determined using ΔG° based on the Gibbs-Helmholtz law and van´t Hoff equation (ΔG°= -RTlnK). Using this fraction value, the amount of unfolded protein at [D] = 0 (Table 1) can be calculated.

Chemical denaturation curve of a mAb

Figure 1. Chemical denaturation curve of a mAb, analyzed by detecting changes in the fluorescence emission ratio using the Prometheus NT.Plex. The fit was performed using a three-state unfolding model.

Table 1. Calculated values illustrating the correlation between ΔG° and the fraction of denatured protein at [D] = 0.

Fraction Denatured

ΔG°(kJ/mol)

0.1

5.4

0.01

11.2

0.001

16.8

1E-4

22.5

1E-5

28.1

1E-6

33.7

1E-7

39.3

1E-8

44.9

1E-9

50.5

1E-10

56.1

1E-11

61.7

1E-12

67.4

1E-13

73.0

1E-14

78.6

The protein stability and ΔG° can be affected by different aggregation mechanisms. By measuring the ΔG° at various protein concentrations, extra information about the aggregation mechanism can be inferred, as aggregation is influenced by the overall protein concentration. There can be three different potential outcomes for this method (Rizzo et al, 2015):

  1. ΔG° is independent of protein concentration, indicating the absence of intermolecular interactions and thus aggregation propensity
  2. ΔG° increases with increasing protein concentrations, meaning that the protein’s folded state is stabilized by “native-state”-aggregation
  3. ΔG° decreases with increasing protein concentration, meaning that the amount of unfolded state is increased by irreversible “denatured-state” aggregation. Thermal unfolding can also be used to accelerate the irreversible “denatured-state” aggregation in temperature ramps. This aggregation can then be determined using the Prometheus NT.Plex with aggregation detection optics, thereby directly obtaining insights into conformational and colloidal stability of proteins.

Here, proof-of-concept chemical denaturation experiments were conducted with lysozyme, which is described in detail in terms of aggregation pathways. In addition, ΔG°app, aggregation onset temperatures (Tagg), and unfolding transition temperatures (Tm) of a mAb in different formulations were analyzed.

The results were then compared with monomer content and turbidity over time as evaluated by HPSEC for long-term stability data. To perform this activity, automated liquid handling was used in conjunction with automated measurement execution by the Prometheus NT.Plex and capillary-chip filling through the NT.Robotic Autosampler.

The results demonstrate that the combination of chemical and thermal denaturation approach allowed for the determination of the formulation that has the best long-term stability.

About CytoSMART

Cytosmart Technologies BVCytoSMART Technologies BV, based in The Netherlands, develops smart solutions for live-cell imaging in cell culture laboratories. The company was founded in 2012 by a group of researchers at the Eindhoven University of Technology, who were frustrated about the lack of inexpensive small imaging systems to support biological cell culture.

Live-cell imaging solutions have remained prohibitively large, complex and very expensive, especially when comparing them to smaller and more cost-effective house-hold imaging systems such as photo cameras and smartphones.

Inspired by recent advances in optics, microelectronics and information technology, the founders of CytoSMART believed that it was time to create new compact imaging systems for biological cell culture.

  • Systems that are small, easy to use and available to everyone.
  • Systems that make advanced live-cell imaging and image analysis possible for all types of cell culture and enable researchers to get a better grip on the complexity of biological cell culture.  
  • Systems that can measure and analyze data in almost real-time. Allowing researchers to act immediately, for better and more reproducible results with cell culture.

The Products

CytoSMART offers the following products for time-lapse live-cell imaging and cell counting:

  • The CytoSMART Lux2, a small, easy-to-use and incubator proof microscope for live-cell imaging.
  • The CytoSMART Exact, an automated cell counter for mammalian cells.
  • The CytoSMART Omni, a compact automated system for full 96 well live-cell imaging inside an incubator

The Support

If you have any question regarding a CytoSMART product, please visit our website at www.cytosmart.com or contact us via email at: [email protected].

If you require technical assistance, our support team is available to answer your phone-call, email or live-chat during weekly office hours (Central European Time, UTC/GMT +1 hr).

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Last updated: Dec 20, 2016 at 10:50 AM

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