Effect of Polyanions on the Structure and Stability of Repifermin™ (Keratinocyte Growth Factor-2)

Derrick, Tiffany; Grillo, Adeola O.; Vitharana, Samadhi N.; Jones, LaToya S.; Rexroad, Jason; Shah, Ambarish; Perkins, Melissa D.; Spitznagel, Thomas M.; Middaugh, C. Russell

doi:10.1002/jps.20797

Cited by 24 publications

(40 citation statements)

References 28 publications

(37 reference statements)

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“…Stoichiometric ratios of 500 -780 (protein : polyanion) were found in the precipitate in the pH range of 1.0 -3.5. A variety of polyanions (e.g., heparin, SOS, and inositol hexaphosphate) increased the extent of aggregation of KGF -2 above 40 ° C, although the thermal unfolding temperature was increased by approximately 9 -15 ° C. 128 The observed differences in aggregation behavior in the presence of polycations/polyanions could also be a result of a concentration -dependent effect. 200 …”

Section: Polycations/polyanionsmentioning

confidence: 97%

“…67 A further increase in temperature above the T m often leads to a more rapid aggregation. Examples include KGF -2 (fi broblast growth factor [FGF] -10), 128 lysozymes, 129,130 BSA, 131 holo -α -lactalbumin, 121 recombinant factor VIII, 132 and β -Lg and κ -CN. 47 When the aggregation process is composed of two distinct steps, nucleation and growth, temperature may affect each step to a different degree.…”

Section: Effect Of High Temperature On Protein Aggregationmentioning

confidence: 99%

“…Because of this, a higher T m does not necessarily mean a lower tendency in protein aggregation. 137 For example, Derrick et al 128 found that a variety of polyanions (e.g., heparin, sucrose octasulfate [SOS], and inositol hexaphosphate) increased the T m of KGF -2 ( T m = 37 ° C) by approximately 9 -15 ° C, but above 40 ° C, aggregation occurred to a greater extent in the presence of these polyanions. Changing the pH of a model monoclonal antibody (mAb) (IgG 2 ) solution from 4.0 to 9.0 increased the thermal stability with more compact conformations, but the rate of aggregation increased at 37 ° C. 138 In reality, changing the experimental conditions can dramatically change the aggregation tendency of a thermally unfolded protein.…”

Section: Melting Temperature ( T M ) and Protein Aggregationmentioning

confidence: 99%

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External Factors Affecting Protein Aggregation

Wang

Speaker

2010

Aggregation of Therapeutic Proteins

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The behavior of biotherapeutic proteins is signifi cantly different from small molecule drugs both in liquid and solid states, especially in terms of physical stability. One such property is the high tendency of protein molecules to aggregate under a variety of conditions. The aggregation tendency is arguably the most common and troubling manifestation of protein instability during the development of protein biotherapeutics. 1 Protein aggregates usually exhibit either reduced or, in many cases, no biological activity. 2 -5 A clear correlation was established between loss of recombinant factor VIII (rFVIII ) and its degree of aggregation as shown in Fig. 4.1 . 6 To achieve effective prevention or inhibition of protein aggregation, it is of paramount importance to understand all the possible factors that affect or control the protein aggregation process.Many factors have been identifi ed and reported in the literature affecting the process and rate of protein aggregation. These factors can be roughly divided into two major categories: internal (protein structure related) and external (protein environment related). Chapter 3 in this book has focused extensively on factors that belong to the fi rst category. This chapter focuses on the external factors that affect the process and rate of protein aggregation. To facilitate discussion, these external factors are further divided into the following sections: (1) temperature; (2) solution conditions and composition (pH, buffer type and concentration, ionic strength, excipients and level, protein concentration, metal ions, denaturing and reducing agents, impurities, organic solvents, containers/closures, sources of proteins, sample treatment, and analytical methodologies); (3) processing steps (fermentation/expression,

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Section: Polycations/polyanionsmentioning

confidence: 97%

Section: Effect Of High Temperature On Protein Aggregationmentioning

confidence: 99%

Section: Melting Temperature ( T M ) and Protein Aggregationmentioning

confidence: 99%

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External Factors Affecting Protein Aggregation

Wang

Speaker

2010

Aggregation of Therapeutic Proteins

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“…Middaugh et al reported that heparin enhanced the thermal stability of human fibroblast growth factor 1 (FGF1) and protected it from low-pH unfolding 29 and that it elevated the temperature of aggregation of keratinocyte growth factor, (KGF)-2, at near-physiological pH and I. 30 Stabilization by heparin of human growth hormone with respect to interfacial denaturation was reported by Zamiri. 31 Wen et al observed that heparin and dextran sulfate stabilize both FGF and KGF by preferential binding to the native state, 32 while Chong et al found that heparin and other anionic polymers stabilize aFGF with respect to aggregation at elevated temperature and neutral pH.…”

Section: Introductionmentioning

confidence: 97%

Suppression of Insulin Aggregation by Heparin

et al. 2008

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The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH-and ionic strength (I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH ) pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I ) 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.

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“…Although conformational stability is generally a good indicator of protein stability in the context of non-native aggregation, it provides an incomplete picture of the downstream processes contributing to aggregation, e.g., the association of aggregation-prone intermediates into higher-order species (15). Moreover, there are instances where unfolding does not necessarily correlate with the propensity towards aggregation, and there are examples of proteins that unfold reversibly (16,17). Here, we report a novel high-throughput approach using ThT (to follow progression of amyloid forming aggregates) in parallel with SYPRO Orange (to follow protein unfolding) to rank-order formulation conditions.…”

Section: Introductionmentioning

confidence: 99%

Orthogonal High-Throughput Thermal Scanning Method for Rank Ordering Protein Formulations

et al. 2013

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Abstract. A high-throughput thermal-scanning method to rank-order formulation conditions for therapeutic proteins is described. Apparent transition temperatures for unfolding and aggregation of four different proteins are determined using the dyes SYPRO Orange and thioflavin T (ThT) under a variety of buffer conditions. The results indicate that the ThT-based thermal scanning method offers several advantages over the previously described SYPRO Orange-based thermal scanning and allows rapid rank ordering of solution conditions relevant toward long-term storage of therapeutic molecules. The method is also amenable to high protein concentration and does not require sample dilution or extensive preparation. Additionally, this parallel use of SYPRO Orange and ThT can be readily applied to the screening of mutants for their unfolding and aggregation propensities.

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Effect of Polyanions on the Structure and Stability of Repifermin™ (Keratinocyte Growth Factor-2)

Cited by 24 publications

References 28 publications

External Factors Affecting Protein Aggregation

External Factors Affecting Protein Aggregation

Suppression of Insulin Aggregation by Heparin

Orthogonal High-Throughput Thermal Scanning Method for Rank Ordering Protein Formulations

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