Developing Biologics Tablets: the Effects of Compression on the

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Developing Biologics Tablets: the Effects of Compression on the Structure and Stability of Bovine Serum Albumin and Lysozyme Yangjie Wei, Chenguang Wang, Bowen Jiang, Changquan Calvin Sun, and C. Russell Middaugh Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01118 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Molecular Pharmaceutics

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Developing Biologics Tablets: the Effects of Compression on

2

the Structure and Stability of Bovine Serum Albumin and

3

Lysozyme

4 5 6

Yangjie Wei,†, ⊥ Chenguang Wang,‡, ⊥ Bowen Jiang §,∥ Changquan Calvin Sun,*, ‡ C. Russell

7

Middaugh *, †

8 9 10

† Department

11

of Pharmaceutical Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA

12

‡

Pharmaceutical Materials Science and Engineering Laboratory, Department of

13

Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota

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55455, USA

15 16

§

Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201, USA

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ACS Paragon Plus Environment

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Oral administration is advantageous compared to the commonly used parenteral

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administration for local therapeutic uses of biologics or mucosal vaccines since it can

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specifically target the gastrointestinal (GI) tract. It offers better patient compliance, even

25

though the general use of such a delivery route is often limited by potential drug degradation

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in the GI tract and poor absorption. Using Bovine Serum Albumin (BSA) and lysozyme as

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two model proteins, their solid-state properties, mechanical properties and tabletability, as

28

well as effects of pressure, particle size, and humidity on protein degradation were studied. It

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was found that BSA and lysozyme are highly hygroscopic and their tablet manufacturability

30

(powder caking, punch sticking, and tablet lamination) is sensitive to the humidity. BSA and

31

lysozyme exhibited high plasticity and excellent tabletability and remained amorphous at

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high pressure and humidity. As for protein stability, lysozyme was resistant to high pressure

33

(up to 300 MPa) and high humidity (up to 93%). In contrast, BSA underwent aggregation

34

upon compression, an effect that was more pronounced for smaller BSA particles. High

35

humidity accelerated the aggregation of BSA during incubation, but it did not further

36

synergize with mechanical stress to induce protein degradation. Thus, compression can

37

potentially induce protein aggregation, but this effect is protein dependent. Therefore,


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strategies (e.g., the use of excipients, optimized manufacturing processes) to inhibit protein

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degradation should be explored before their tablet dosage form development.

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KEYWORDS: protein tablet, protein structure, protein stability, aggregation, lysozyme, BSA


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INTRODUCTION

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Recent decades have witnessed the success of numerous biologics as highly effective

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drugs.1,

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forms rather than orally administrated due to their instability in acidic gastric fluid,

45

enzymatic digestion in the intestine lumen, and low permeation through intestinal

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epithelium.3 However, oral administration of biologics possesses significant advantages,

47

including better patient compliance and delivery of therapeutics to certain targets for

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treating gut diseases. Major joint efforts between academia and industry have been made to

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overcome the biologic barriers in the gastrointestinal (GI) tract to bring oral biologics into

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clinical use.3-7 The recent clinical success of a GLP-1 analogue oral tablet in late stage

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development suggests that the first orally delivered biologic product may soon be available.8-

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10

2

Therapeutic macromolecules are commonly formulated into parenteral dosage

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Tablets remain the most widely accepted oral dosage form because of patient compliance,

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manufacturing efficiency, and economy. To manufacture tablets of biologics, three critical

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process steps are involved. Firstly, drying of proteins/peptides into solids, which is currently

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achievable at large scale by lyophilization, spray drying, or foam drying.11-14 The aseptic

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spray drying technique also enables powder engineering of biologics.15-19 Secondly,

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compression of biologics, with or without excipients, into tablets. The compression of

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biologics is a relatively new but critical unit operation with the potential to cause physical

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degradation of proteins/peptides due to exposure to mechanical, thermal, and shear


4

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stresses.20,

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ingredient (API) from degradation in the upper GI tract. The polymeric enteric coating has

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been well established for lower molecular weight APIs but only successfully applied to

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biologics in a few cases (e.g., adenovirus vaccine tablets).22-25

21

Finally, the enteric coating of tablets to protect the active pharmaceutical

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Among these three steps, which all contribute to the successful development of protein

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tablets, the compression process has not been systematically investigated. It would seem that

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a joint effort from material science, process engineering, and protein chemistry might

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facilitate a mechanistic understanding of possible protein changes during compression.

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Klukkert et al. observed a loss of trypsin activity, perturbed secondary structure and reduced

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refolding following compression.20 Picker et al. concluded that the enzymatic activity loss of

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alpha-amylase was dependent on their subtypes and can be obviated by adding excipients,

72

such as microcrystalline cellulose.26

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The tableting performance of proteins is expected to depend on their solid state form,

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particle morphology, and mechanical properties.27 Although important, there is a lack of

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prior knowledge concerning protein mechanical properties and tabletability. With the use of

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a nanoindenter, mechanical properties, such as hardness and elastic modulus, can be

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determined using small amounts of material. This information can be used to predict

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tableting performance and to guide excipient selection.27, 28

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In this work, two model proteins bovine serum albumin (BSA) and lysozyme as were

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selected due to their distinct molecular properties, where lysozyme has a small molecular

81

size and high stability and BSA has a high molecular weight, lower stability, and


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hydrophobic binding sites. The two proteins were fully characterized based on their solid-

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state properties, particle morphology, hygroscopicity, mechanical properties, and

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tabletability. After compression, the protein solids were reconstituted in PBS buffer and

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characterized using orthogonal biophysical and analytical techniques. The effects of particle

86

size, moisture content, and compression pressure on protein structure and stabilities were

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assessed.

88 89

MATERIALS AND METHODS

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Materials and sample preparation. BSA and lysozyme lyophilized powders were

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purchased from Sigma-Aldrich (St. Louis, MO). To study the effect of size on stability,

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protein powders with various particle size ranges (500 μm) were

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obtained using standard sieves of #60, 45, and 35 mesh (W.S. Tyler Industrial Group, Mentor,

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OH). Each size fraction was compressed under various pressures (10~300 MPa) to form

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tablets using a material testing machine (model 1485; Zwick/Roell, Ulm, Germany) at a speed

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of 2 mm/s using 6 mm diameter flat-faced round tooling. The compression was uniaxial and

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target tablet weight was 20 mg. The protein powders were manually filled into the die and three

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tablets were prepared under each condition. The effects of relative humidity (RH) on the

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degradation of protein were also investigated by pre-equilibrating protein powders (< 250 μm

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for BSA, 250~355 μm for lysozyme) for one month in various RH chambers over saturated

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aqueous solution of salts, magnesium chloride (RH 32%), magnesium nitrate (RH 52%),

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sodium chloride (RH 75%), and potassium nitrate (RH 93%) at 25°C prior to compression at


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100 or 200 MPa. The use of extremely high RHs was mainly to gain a more comprehensive

104

understanding of RH effects regardless of relevance to real-life settings. However, high

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humidity could arise during wet granulation or exposure to high RH environment during

106

storage.

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Protein tablets were reconstituted in PBS buffer (10 mM phosphate, 150 mM NaCl, pH 7.4)

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to achieve a stock concentration of approximately 50 mg/mL. Reconstituted samples were

109

suitably diluted and subjected to appropriate biophysical analyses. Uncompressed protein

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powders were used as controls throughout this study. A detailed workflow listing the

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compression variables investigated and biophysical techniques employed is present in Table

112

1.

113 114

Table 1. Model proteins tested, variables investigated, and biophysical techniques utilized to

115

characterize protein samples in this study. For each particle size fraction, samples compressed at

116

all pressures were characterized unless otherwise noted. Techniques applied to only characterize

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tablets compressed at highest pressures are marked * (BSA) and # (lysozyme). Model protein

BSA

Particle size fraction (μm)

> 500

355~ 500

Pressure (MPa)

10, 25, 50, 100, 200, 250, 300 *#

Characterization

Lysozyme 250~ 355

< 250

> 500

355~500

10, 50, 100, 200

< 250

250~355

10, 50, 100, 200, 300 #

FT-IR , Raman, Fluorescence, DSC , DLS, SLS/OD #

# 350nm

#

, SEC ,

Enzymatic assay


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Humidity study Protein sample RH

BSA (< 250 μm) 32%

52%

75%

Lysozyme (250~355 μm) 93%

32%

52%

Pressure (MPa)

0, 100, 200

Characterization

Fluorescence, DSC , DLS, SEC

#

75%

93%

#

118 119 120

Polarized light microscopy (PLM). Protein samples were observed under a polarized light

121

microscope (Eclipse e200; Nikon, Tokyo, Japan), equipped with a DS-Fi1 microscope digital

122

camera for capturing digital images.

123

Powder X-ray Diffractometry (PXRD). Powders were analyzed using an X-ray

124

diffractometer (PANalytical X’pert pro, Westborough, MA) with Cu Kα radiation (1.54056

125

Å), with two-theta pre-calibrated using a silicon standard. Samples were scanned from 5 to

126

35° two theta with a step size of 0.017° at 1 s/step. The tube voltage and amperage were

127

maintained at 40 kV and 40 mA, respectively.

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Dynamic Water Vapor Sorption Isotherm (DVS). A sieve cut between #120 and #60 mesh,

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i.e., 125–250 μm size fraction, of each protein powder was used for the DVS study. Water

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sorption and desorption profiles of the materials were obtained by using an automated vapor

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sorption analyzer (DVS 1000, Surface Measurement Systems Ltd., Alperton, Middlesex, UK)


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at 25 °C. The nitrogen flow rate was 50 mL/min. Samples were equilibrated at each step with

133

the equilibration criteria of either dm/dt ≤0.002% with a minimum equilibration time of 0.5

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h or maximum equilibration time of 6 h. Once one of the criteria was met, the RH was

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changed to the next target value from 0 to 95% with a step size of 5% RH.

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Nanoindentation. A triboindenter I980 (Hysitron, Minneapolis, MN) equipped with a

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three-sided pyramidal Berkovich diamond tip was employed to determine mechanical

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properties (T = 24℃, RH ~50%). Before the experiment, tip area function was calibrated with

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a series of indents at various contact depths on fused silica. Large protein samples were

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mounted onto a glass slide using superglue and the slide was fixed on the stage by vacuum.

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An area of 50×50 µm2 was scanned using the indentor tip in contact mode with the protein

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surfaces using a 2 µN force. An area with roughness less than 20 nm was used for the

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indentation study. Experiments were conducted with maximum tip penetration depth of

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1000 nm under two displacement control modes: 1) single loading and unloading; and 2)

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multiple partial loading and unloading. There are three segments in each cycle: 5 s linear

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loading, followed by 10 s holding at peak displacement and 5s unloading. The linear

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unloading part of the force-displacement curve was used to extract the elastic contact

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stiffness (S, µN/nm), which was used to calculate reduced elastic modulus (Er) using Eqn. (1).

149

The Poisson’s ratios of 0.3 and 0.07 for protein samples (ν) and indenter (νi), respectively,

150

were assumed for calculating the Young’s modulus (E, GPa) of samples using Eqn. (2), using

151

1141 GPa for the elastic modulus of the diamond indenter (Ei). The contact hardness (Hc,


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GPa) was calculated using peak load force (Pmax, µN) divided by residual contact area (A(hc),

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µm2) using Eqn. (3), where A(hc) was calculated using Eqn. (4), according the Oliver–Pharr

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method.29 𝜋𝑆 𝐴(ℎ𝑐)

155

𝐸𝑟 = 2

156

1 𝐸𝑟

157

𝐻𝑐 = 𝐴(ℎ𝑐)

158

ℎ𝑐 = ℎ𝑚 ―

=

(1 ― 𝑣2) 𝐸

+

(1) (1 ― 𝑣𝑖2)

(2)

𝐸𝑖

𝑃𝑚𝑎𝑥

(3) 3𝑃𝑚𝑎𝑥

(4)

𝑆

159

Where hm and hc are the recorded maximum and residual contact displacement (µm),

160

respectively.

161

Tabletability. To test the effects of size and compression pressure on tablet tensile strength,

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two size factions (250~355 µm and >500 µm) of BSA and lysozyme powders were compressed

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at 50, 100, 150, 200, 250, 300, and 350 MPa. Protein tablets were allowed to relax under the

164

ambient environment for 1 h before the diametrical breaking force was measured using a

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texture analyzer (TA-XT2i; Texture Technologies Corporation, Scarsdale, New York). Tablet

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tensile strength was calculated from the breaking force and tablet dimensions following Eqn

167

(5).30

168

2𝐹

𝑇𝑒𝑛𝑠𝑖𝑙𝑒 𝑠𝑡𝑟𝑒𝑛𝑔𝑡ℎ = 𝜋𝐷ℎ

(5)


10

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169

Where F, D, and h are the breaking force (F, N), tablet diameter (D, mm), and tablet

170

thickness (h, mm), respectively.

171

Fourier Transformation Infrared Spectroscopy (FTIR). FTIR spectra of protein samples

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(at 50 mg/mL) were collected using a Tensor-27 FTIR spectrometer (Bruker, Billerica, MA) at

173

25 oC. The Mercury cadmium telluride (MCT) detector was cooled using liquid nitrogen for

174

at least 30 min prior to use. A total of 256 acquisitions were made at a resolution of 2 cm-1.

175

Raw spectra were processed using OPUS V6.5 (Bruker, Billerica, MA). Atmospheric

176

compensation (water vapor and CO2) and baseline adjustment were performed. The amide I

177

band (1600~1700 cm-1) was normalized. Second derivatives of the processed FTIR spectra

178

were obtained using a window size of 9.

179

Intrinsic fluorescence. The intrinsic fluorescence of reconstituted protein samples (at 10

180

mg/mL) were measured using a steady-state fluorescence plate reader previously described 31.

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Samples were excited at 295 nm (> 95% tryptophan), and their emission spectra were

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collected from 310 to 400 nm using an acquisition time of 0.25 s. A thermal ramp was set

183

from 10 to 90 oC with an increment of 2.5 oC per step and an equilibration time of 120 s. The

184

mean spectral center of mass (MSM) peak positon of the spectra (Eqn. 6) was derived and

185

plotted against temperature to generate melting curves. Melting temperatures were taken as

186

the maximal point of melting curves’ first derivative using Origin software (OriginLab

187

Corporation, Northampton, MA).


11


188 189

MSM peak position =

∫𝐹(𝜆)𝜆𝑑𝜆 ∫𝐹(𝜆)𝑑𝜆

Page 12 of 42

(6)

Where F (λ) is fluorescence intensity at a specific wavelength, λ.

190

Raman spectroscopy. Raman spectroscopy was performed using a Zetasizer Helix (Malvern

191

Instruments, Columbia, MD) equipped with a 785 nm laser (~280 mW). BSA and lysozyme

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samples (at 50 mg/mL) were loaded into a micro-cuvette (Malvern Instruments) and

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measured at 25 oC. Raman spectra were collected from 1800~400 cm-1 with a 4 cm-1

194

resolution using an acquisition time of 20 sec and a total of 10 acquisitions per run. Raman

195

spectra were further processed and analyzed using the Zetasizer Helix Analyze software

196

(Malvern Instruments). Raw spectra were normalized according to the phenylalanine peak at

197

~1000 cm-1. The secondary structure content of protein samples was derived using a

198

multivariate analysis (MVA) of the Raman spectra. This MVA approach incorporates a rather

199

broad spectral range (~990 to 1730 cm-1). The secondary structure content was derived

200

according to a partial least squares regression model.

201

Differential scanning calorimetry (DSC). A VP-Capillary micro-calorimeter (Malvern,

202

UK) was used to investigate the overall conformational stability of reconstituted BSA and

203

lysozyme. Protein solution at 1 mg/mL were stored in a 96-well plate kept in a 5 oC chamber,

204

and 400 μL of sample was injected. Samples were scanned from 10 to 100 ᵒC using a scan rate

205

of 2 ᵒC/min. DSC thermograms were buffer subtracted, normalized using the molar

206

concentration of the proteins, and fit to a non-two-state model to calculate melting

207

temperatures using Origin software (OriginLab Corporation, Northampton, MA).


12

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208

Static light scattering (SLS). An Optim 1000 instrument (Avacta Innovative Analysis, UK)

209

was used to measure the static light scattering of protein samples. Protein samples (at 1

210

mg/mL) were added into an Optim micro-cuvette array (Avacta Innovative Analysis) and

211

then loaded onto a temperature controlled sample plate. Temperature was ramped from

212

10~100 ᵒC with a step size of 2.5 oC and an equilibration time of 120 s. Scattering signals

213

were measured using a 473-nm laser and an acquisition time of 200 ms.

214

Optical Density at 350 nm (OD350nm). A UV-Visible spectrophotometer (Cary 100, Varian

215

medical Systems, Inc., Palo Alto, California) was employed to monitor the OD350nm of protein

216

samples upon thermal stress. Samples were placed in 1x1 cm quartz cuvettes. Temperature

217

was ramped from 10 to 90 oC with an increment of 2.5 oC per step and an equilibration time

218

of 120 sec.

219

Dynamic light scattering (DLS). DLS was performed using a DynaPro plate reader (Wyatt

220

Technology, Santa Barbara, CA). Protein samples (at 20 mg/mL) were loaded into a black

221

clear bottom 384-well plate (Corning Incorporated, Corning, NY). The plate was centrifuged

222

at 2000 rpm for 1 min to remove air bubbles. Samples were equilibrated at 25 ᵒC for 10 min

223

prior to measurement. Each sample was analyzed using 5 acquisitions of 10 sec each time.

224

Translational diffusion coefficients (Dt) were derived by fitting the autocorrelation function

225

using a cumulant analysis. Hydrodynamic radii (Rh) were further calculated using the Stokes-

226

Einstein equation (Eqn. 7), and intensity-averaged size was reported.


13


𝑘𝑇

227 228

Dt = 6𝜋𝜂𝑅ℎ

Page 14 of 42

(7)

Where k, η, and T are the Boltzmann constant, the viscosity, and temperature, respectively.

229

Size exclusion chromatography (SEC). SEC was carried out using an HPLC instrument

230

(Shimadzu, Kyoto, Japan) equipped with a photo diode array detector and a column oven. A

231

mobile phase consisting of a 0.5 M sodium phosphate buffer (pH 6.8) was used. Twenty

232

microliters of samples (at 1 mg/mL) were injected into a TSKgel SWXL guard column (6.0

233

mm x 40 mm) followed by a TSKgel G3000SWXL SEC column (7.8mm x 300 mm) at a flow

234

rate of 0.7 mL/min for a total run time of 30 min. Column oven temperature was set at 30 oC.

235

Chromatograms detected at 214 nm were analyzed using LC station software (Shimazdu,

236

Kyoto, Japan).

237

Enzymatic activity assay. Enzymatic activities of lysozymes were measured using the

238

EnzChekTM lysozyme assay kit (Invitrogen™, Carlsbad, CA). Suitably diluted lysozyme

239

samples were mixed at a 1:1 volumetric ratio with a lysozyme substrate, Micrococcus

240

lysodeikticus, suspension (at 50 μg/mL), labeled with fluorescein. The mixture was incubated

241

at 37 ᵒC for 30 min in the dark. The fluorescence intensity was measured in a fluorescence

242

plate reader (Corning Incorporated, Corning, NY) using an excitation/emission wavelength

243

of 485/530 nm.

244

RESULTS


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245

Solid-state properties. The absence of intense peaks in the PXRD patterns and lack of

246

birefringence confirmed that the as-received BSA and lysozyme powders were amorphous

247

(Figure 1A). Since both BSA and lysozyme were flaky with size ranging from 50 µm to 3 mm

248

as measured by microscopy, sieved samples were used to minimize particle size

249

heterogeneity.

250

The hygroscopicity of these two proteins was measured to assist the better understanding

251

of changes in their physical or chemical properties induced by RH variation, which can

252

guide appropriate handling during manufacturing and weight corrections in assay methods.

253

Both proteins continuously gained weight when exposed to progressively increasing RH up

254

to 95% (Figure 1B). The two sorption curves were superimposable over the 0-50% RH range.

255

At higher RHs (55-95%), the BSA tended to absorb more water than lysozyme. The weight

256

gained for BSA was 19.9% (w/w) and for lysozyme was 16.4% at RH 80%. Thus these two

257

proteins are very hygroscopic materials based on the criteria in the European

258

Pharmacopoeia.32 The high hygroscopicity is in agreement with their amorphous nature,

259

where both surface adsorption (typically < 0.5 %) and absorption of water can occur.33 Thus,

260

both surface and bulk properties of these proteins are expected to depend on RH.

261

RH effects could result in particle morphology changes, powder caking, punch sticking,

262

and tablet lamination, which are detrimental to processes control and cause

263

manufacturability issues. The BSA and lysozyme powders exhibited distinct behaviors when

264

stored at high RHs. The BSA powder caked at 75% and 93% RH, which is unfavorable for


15


Page 16 of 42

265

powder handling during commercial manufacturing. This is accompanied by significant

266

shrinkage of BSA particles at 75% and 93% RH (Figure 1C). On the other hand, the lysozyme

267

powder did not cake even at RH 93% and particle morphology did not undergo observable

268

changes. However, lysozyme stored at RH 75% and 93% exhibited severe punch sticking

269

and tablet lamination during the powder compression. Therefore, the powder properties of

270

lysozyme are also modified by water. Interestingly, the compressed BSA tablets did not have

271

any sign of sticking and lamination. BSA tablets prepared at ≥200 MPa using powder stored

272

at RH 93% became translucent and showed some birefringence under PLM (Figure 1A). It

273

has been shown that the compression and water may induce amorphous to crystalline

274

transformations of small molecule drugs during the tablet manufacturing process.34,

275

However, the crystallization of BSA was excluded by the absence of crystalline peaks in

276

PXRD patterns of the tablet, the birefringence indicates residual stresses in the tablet. In

277

summary, the proteins remained amorphous even at the highest pressure (300 MPa) and

278

humidity (93%), but both proteins undergo significant changes in powder properties,

279

including caking, punch sticking, and tablet lamination, when the humidity is ≥75%.

35


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(A)

(B)

(C)

280 281

Figure 1. Characterization of BSA and lysozyme (A) PXRD patterns of powder and tablet

282

along with some representative PLM images. (B) Moisture sorption isotherms (100-250 µm

283

fraction). 3) Morphology of two proteins at different storage humidities viewed by PLM (the

284

width of each image is 3.2 mm).

285 286

Mechanical properties and tabletability. Mechanical properties play a central role in tablet

287

manufacturing and product performance.36 Although the mechanical properties of small


17


Page 18 of 42

288

molecule drugs and pharmaceutical excipients have been routinely reported, mechanical

289

properties of pharmaceutical proteins have rarely been explored. We are unaware of

290

systematical characterization of mechanical properties of both protein particles and bulk

291

powders. Hence, in this study, we have determined the E and H of BSA and lysozyme using

292

nanoindentation. Before the indentation, the protein’s surface was mapped using a sharp

293

Berkovich tip and an area with roughness less than 20 nm was chosen for indentation. No

294

fracture was observed after the indentation for both protein samples, indicating neither

295

protein is brittle. The load – depth curves of BSA and lysozyme proteins are smooth without

296

any ‘pop-in’ events during loading or ‘pop-out’ during unloading (Figure 2A). This suggests

297

an absence of molecular slip planes often observed in small molecule crystals or phase

298

change.37 At the same indentation force, BSA showed less penetration depth (Figure 2A).

299

Therefore, the BSA (E = 4.09±0.43 GPa; H = 0.13±0.02 GPa) is slightly less stiff and more

300

plastic than lysozyme (E = 5.25±0.46 GPa; H = 0.19±0.03 GPa). The multiple partial loading

301

and unloading nanoindentation results led to E and H values comparable to those from the

302

single loading and unloading experiments, implying negligible impact by indentation depth.

303

The measured H value is 0.15±0.02 GPa for lysozyme and 0.14±0.03 GPa for BSA (Figure 2B)

304

with depth ranging from ~40 to ~900 nm.


18

Page 19 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


305 306 307

Figure 2. Representative load–depth curves of BSA and lysozyme using A) single loading and

308

unloading; B) multiple partial loading and unloading methods using a nanoindentation test

309

(RH ~ 50%).

310 311

The median of reported 283 E and H values of biomaterials are 8.89 GPa (ranging 8.5×10-6

312

to 142.5 GPa) and 0.49 GPa (ranging 1.3×10-6 to 11.4 GPa), respectively.38 For pharmaceutical

313

crystals, the median of 222 reported E and H values are 11.43 GPa (ranging 0.27 to 46.8 GPa)

314

and 0.43 GPa (ranging 1.0×10-3 to 1.8 GPa), respectively.39 Therefore, both BSA and

315

lysozyme showed E and H values lower than the majority of inorganic crystals, metals,

316

organic crystals, and biomaterials.38-41 The softness of these two proteins is consistent with

317

their amorphous nature and weaker intermolecular interactions than above-mentioned other

318

classes of materials. Their mechanical properties are more comparable to the solid dispersion

319

of Kollidon VA64 with clotrimazole (E = 4.3 - 6.1 GPa and H = 0.05 - 0.33 GPa in the 18% -


19


Page 20 of 42

320

49% RH range),42 and acetaminophen (H = 0.17 - 0.2 GPa at 35% RH).43 The low H values

321

of BSA and lysozyme suggest that plastic deformation is the dominating deformation

322

mechanism under pressure, which generally favors tablet formation through effectively

323

increasing the inter-particle bonding area.44, 45

324

Based on the bonding area - bonding strength interplay model,44 the high plastic material could

325

easily reach and maintain the highest tablet tensile strength plateau compared to the brittle

326

material. A plateau is observed when further increase in pressure does not lead to larger bonding

327

areas among particles. In addition, it is expected that increases of inter-particle bonding area

328

through particle size reduction is not an effective way of increasing the tablet strength of BSA

329

and lysozyme since the bonding area is not the limiting factor. At RH 42%, both BSA and

330

lysozyme form tablets with tensile strength higher than 2 MPa at 200 MPa (Figure 3), which

331

may be attributed to their high plasticity.46 The high plasticity also explains the small effects

332

of particle size on tabletability

333

(Figure 3). If needed, both BSA and lysozyme can be compressed into sufficiently strong

334

tablets without using any excipients given their good tabletability. Punch sticking and tablet

335

lamination did not occur during compression at 42% RH.

47

and the plateau in tabletability profiles above 200 MPa


20

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336 337

Figure 3. Effects of particle size on the tabletability of BSA and lysozyme at ~42% RH (n=1).

338 339

The effects of compression on protein secondary and tertiary structures after

340

reconstitution. Mechanical stresses may alter the structure of a protein in the solid state. 20 A

341

therapeutically relevant question for a tablet of potential protein drug is whether such

342

structural alterations, if they exist, persist after protein tablets are dissolved in solution. If

343

these structural alterations in the solid state disappear in solution, they are reversible and

344

may not have detrimental effects on the drug’s efficacy. In contrast, irreversible alterations

345

can result in a loss in drug potency and other issues. Therefore, the BSA and lysozyme tablets

346

were reconstituted and characterized their structural properties in solution.

347

The secondary structure of reconstituted protein samples were investigated using FTIR.

348

The amide I band (1600 to 1700 cm-1) primarily originates from the C=O vibration and,

349

therefore, reflects the backbone conformation and hydrogen bonding patterns (i.e.,


21


Page 22 of 42

350

secondary structure of a protein). The amide I spectra of compressed protein samples

351

overlapped with their control samples, suggesting similar secondary structure before and

352

after compression (Figures 4A1 and 4A2). Since an FTIR amide I spectrum comprises several

353

overlapping absorption peaks, a second derivative analysis of the spectrum is performed to

354

resolve these peaks (Figure S1A). The second derivative spectra of these samples are also

355

indistinguishable from the control samples, further confirming that compressed BSA and

356

lysozyme retained their secondary structures in solution (Figure S1B).

357

Raman spectroscopy measures vibrational modes of the protein backbone and side chains,

358

and therefore provides rich information regarding a protein’s secondary and tertiary

359

structures. The amide I (1630~1680 cm-1) and III (1230~1350 cm-1) bands are often used to

360

probe secondary structure. As shown in Figure 4B1, BSA samples (control and compressed)

361

had identical amide bands, again supporting similar secondary structures suggested by the

362

FTIR analysis. Derived secondary structure contents of these protein samples did not reveal

363

significant differences (Table S1). The same analysis performed for lysozyme samples also

364

illustrates an absence of effects on their secondary structures by high pressures (Figure 4B2

365

and Table S2).

366

Side chain peaks of Raman spectra were then analyzed to study the protein’s tertiary

367

structures upon compression, including the analysis of tryptophan band position (indole ring

368

angle), tyrosine doublet ratio I847/I831 (hydrogen bonding), and the position of the tyrosine

369

peak at 857 cm-1. None of these parameters was significantly altered in compressed BSA or


22

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370

lysozyme relative to the control samples (Tables S1 and S2), suggesting no significant

371

alterations in their tertiary structures.

372

Intrinsic fluorescence is commonly used to probe subtle changes in a protein’s tertiary

373

structure, since the fluorescence of tryptophan residues (often buried inside a protein) is

374

highly sensitive to the polarity of their surrounding environment (e.g., solvent exposure).

375

Upon protein unfolding, tryptophan residues become exposed to more polar environments

376

and this often causes a red shift in fluorescence spectra. Since BSA (66 kDa) and lysozyme (14

377

kDa) contain 2 and 6 spatially distinct tryptophan residues, respectively, the use of

378

tryptophan fluorescence provides a robust method to probe changes in their overall tertiary

379

structures. All BSA and lysozyme samples subjected to compression shared identical

380

fluorescence spectra compared to the control samples (Figures 4C1 and 4C2), confirming that

381

compression did not significantly alter their tertiary structure.


23


Page 24 of 42

382 383

Figure 4. Effects of powder particle size and pressure on the secondary and tertiary structures of

384

reconstituted BSA and lysozyme tablet samples in PBS buffer at 25 oC. FT-IR Amide I band

385

spectra of BSA (A1) and lysozyme (A2) samples. Raman spectra of reconstituted BSA (B1) and

386

lysozyme (B2) tablet samples in PBS buffer at 25 oC. Normalized tryptophan fluorescence (FL)

387

spectra of BSA (C1) and lysozyme (C2). Error bars represent standard errors based on data for

388

three samples.


24

Page 25 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


389

Thermal stability of reconstituted protein tablet samples. Proteins under thermal stress

390

can undergo structural alterations, including significant unfolding accompanied by changes

391

in their fluorescence signals. Plotting a stability indicating parameter derived from raw

392

fluorescence spectra as a function of temperature can be used to assess thermal stability of a

393

protein. Here, the MSM peak position instead of the apparent one (i.e., the position

394

corresponding the maximal fluorescence intensity) was used to construct the melting curves,

395

since the former term is calculated by integration of the whole spectra and offers improved

396

signal-to-noise ratio. BSA samples under thermal stress exhibited a gradual blue shift in their

397

fluorescence spectra, probably indicating an overall decrease in the solvent exposure of

398

tryptophan residues (Figure 5A1). The melting temperatures (Tm) of all of the BSA samples

399

(control and compressed) were found to be approximately 68.5 oC, demonstrating similar

400

thermal stabilities. In contrast, lysozyme showed a slight increase in MSM peak positions as

401

temperature increases, possibly attributable to a thermal quenching effect of solvent exposed

402

tryptophan residues (W62, W63, W123, etc.) (Figure 5A2). A sudden red shift in MSM peak

403

positions was observed upon protein thermal unfolding, suggesting tryptophan residues

404

became more exposed. Compressed lysozyme samples shared similar thermal stability with

405

the control sample, as evidenced by their identical thermal unfolding curves.

406

Differential scanning calorimetry (DSC) is an information-rich technique for studying

407

overall conformational stability of proteins. DSC measures changes in the partial molar heat

408

capacity of a protein at constant pressure (ΔCp) upon thermal perturbation. As shown in


25


Page 26 of 42

409

Figure 5B1, DSC thermograms of BSA samples in PBS buffer showed two major thermal

410

transitions (Tm1 ≈ 64.5 and Tm2 ≈ 79.0 oC). All of the BSA samples tested had identical Tm

411

values (Figure S2A). On the other hand, an overall decrease in the magnitude of BSA

412

thermograms with compression pressure was found (Figure S2B). This suggests that

413

compression induced a higher level of non-native proteins since they usually require less

414

heat to thermally unfold. Unlike BSA, lysozyme in PBS buffer exhibited one thermal

415

transitions with a Tm of 72~73 oC (Figure 5B2 and Table S3). All of the lysozyme samples

416

tested showed overlapping thermograms of similar magnitude. These data clearly indicate

417

that the thermal stabilities of compressed lysozyme samples remained unchanged after they

418

were reconstituted in PBS buffer.

419

A common phenomenon accompanied by thermally induced protein unfolding is protein

420

aggregation. It can be measured by optical density using a conventional UV/Vis

421

spectrophotometer or by static light scattering, since protein aggregates strongly scatter light

422

resulting in increased optical density. Protein aggregation behavior of BSA and lysozyme

423

under thermal stress was monitored using static light scattering and optical density at 350 nm

424

(OD350nm), respectively. No discrepancy was observed in the thermally induced aggregation

425

profiles of BSA or lysozyme subjected to various levels of pressure (Figures 5C1 and 5C2).


26

Page 27 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


426 427

Figure 5. Thermal stability of reconstituted BSA and lysozyme tablets in PBS buffer. Thermal

428

melting curves of BSA (A1) and lysozyme (A2) reflected by temperature-induced alterations in

429

the MSM peak position of fluorescence spectra. Differential scanning calorimetry thermograms

430

of BSA (B1) and lysozyme (B2). Colloidal stabilities of reconstituted BSA (C1, measured by


27


Page 28 of 42

431

static light scattering) and lysozyme (C2, measured by optical density at 350 nm) samples in

432

PBS buffer under thermal stress, respectively. Error bars represent standard errors based on data

433

for three samples.

434 435

Protein aggregation level induced by compression. Compression of proteins in the solid

436

state reduces intermolecular distances and can potentially induce aggregations (reversible or

437

non-reversible). Aggregation is a significant concern for biotherapeutics since it may cause

438

unwanted immunogenicity and a loss of biological activity

439

examined in reconstituted samples using DLS and SEC. Both techniques measure the Rh of a

440

particle. Since DLS intensity signals rapidly increase with the size of a particle, the intensity

441

averaged size results in a value weighted towards the larger end of a size distribution. This

442

makes such parameter quite sensitive for the detection of even a minute level of aggregates.

443

The BSA control sample (at 10 mg/mL) at 25 oC was found to have an Rh of 3.7 ± 0.1 nm

444

(Figure 6A). The Rh of BSA samples subjected to higher pressure became larger. Although

445

such a trend holds for all size ranges of BSA investigated, smaller BSA particles were more

446

prone to compression-induced aggregation. In contrast, all of the lysozymes tested (at 10

447

mg/mL) had Rh values independent of their particle sizes and pressure in a range of 1.9 ~ 2.0

448

nm (Figure 6B), corresponding to monomeric lysozyme 49.

48.

The level of aggregates was

449

Since SEC is one of the most commonly used techniques to quantify protein aggregation, it

450

was used as an orthogonal method to DLS to analyze reconstituted BSA and lysozyme


28

Page 29 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


451

samples. The SEC column used in this study had an ideal separation range of 10 to 500 kDa.

452

Therefore, it is able to readily separate aggregates, if any, formed in BSA (66 kDa) and

453

lysozyme (14 kDa) samples. A significant level of aggregate was detected in the BSA control

454

sample (Figure 6C), which accords with the fact that BSA samples typically contain some

455

level of oligomers. Compressed samples showed more dimer and high molecular weight

456

(HMW) species. BSA samples subjected to higher pressure had a larger decrease in their

457

monomer contents, i.e., more extensive aggregation (Figure 6D). Such a trend is also highly

458

dependent on the particle size of BSA samples. BSA with a smaller particle size was more

459

susceptible to pressure induced aggregation. On the other hand, aggregates are absent in all

460

of the lysozyme samples tested (Figure S3). Overall, the SEC data for both proteins correlate

461

well with their DLS results. Reconstituted BSA samples showed pressure- and particle size-

462

dependent aggregation profiles, while lysozyme did not form a detectable level of irreversible

463

aggregates in all compressed samples.


29


Page 30 of 42

464 465

Figure 6. Aggregation levels of BSA and lysozyme tablet samples upon reconstitution in PBS

466

buffer at 25 ᵒC. Rh of BSA (A) and lysozyme (B) samples investigated by DLS. Representative

467

SEC chromatograms of BSA (C) and the loss of BSA monomer (D). Error bars represent

468

standard errors based on data for three samples.

469 470

Enzymatic activity of compressed lysozyme. A more direct pharmaceutically relevant

471

assay for a potential therapeutic protein tablet is biological activity. The biological activity of

472

lysozyme samples was measured. Lysozyme enzymatically hydrolyzes 1,4-β-linkages


30

Page 31 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


473

between

474

peptideglycans, a key component of a variety of microorganisms’ cell walls. Such enzymatic

475

activity can be quantified using a fluorescently labelled substrate of lysozyme. Lysozyme

476

hydrolysis of this substrate results in a dramatic increase in the fluorescence signal, which is

477

linearly proportional to lysozyme activity within a certain range. Reagents employed in this

478

study can detect lysozyme activity as low as 20 U/mL. All of the stressed lysozyme had

479

similar levels of enzymatic activity to that of the control sample (Figure S4). This result again

480

suggests lysozyme is highly resistant to mechanical stresses.

481

N-acetylmuramic

acid

and

N-acetyl-D-glucosamine

residues

present

in

Effects of humidity on aggregation. Humidity is a critical manufacturing process

482

parameter that affects manufacturability and solid-state stability of drugs.50,

483

effects of RH on the structure and stability of compressed proteins were also investigated.

484

Incubation of BSA powders in all four RH conditions resulted in significant increases (0.6 ~

485

20.5%) in aggregate formation relative to the BSA control sample stored at 4 ᵒC (Figures 7A

486

and S5). Overall, BSA incubated under a higher RH aggregated to a greater extent.

487

Compression induced further aggregation in BSA equilibrated under all of the four RH

488

conditions. This effect did not strongly depend on RH, where increases in BSA aggregates by

489

200 MPa compression pressure at 33% and 93% RH were 2.2 ± 0.1% and 2.5 ± 0.2 %,

490

respectively (Figure 7A). Thus, RH did not synergize with compression in term of BSA

491

aggregation. DLS data of BSA samples (Figure 7B) correlated well with the SEC data. BSA

492

samples incubated at 93% RH exhibited a significant red shift (> 0.5 nm) in the MSM peak

51

Here, the


31


Page 32 of 42

493

positions of their tryptophan fluorescence spectra with the sample subjected to the highest

494

pressure showing the largest blue shift in spectra peak position at 10 oC (Figure 7C). Such a

495

small structural change was not observed for samples incubated under lower RH conditions

496

(33%, 52%, and 75%) (Figure S6). This suggests an alteration of BSA’s tertiary structure post

497

a one-month incubation period at 93% RH, which were further enhanced by higher

498

pressure. BSA samples incubated at 93% RH showed weaker thermal transitions with lower

499

calorimetric area under the curve (AUC) values than the control BSA sample (Figures 7C and

500

7D). This trend suggests that BSA incubated under 93% RH formed more non-native forms,

501

which require less energy to thermally unfold the protein. BSA incubated under lower RH

502

levels (33% and 52%) showed minimal loss of calorimetric AUCs. In contrast, SEC analysis of

503

lysozyme samples did not show any detectable level of aggregates (Figure S7A), and the Rh of

504

lysozyme samples remained unchanged (Figure S7B). All of the stressed lysozyme samples

505

had similar fluorescence emission signals and thermal stability compared to the control

506

samples (Figures S7C and S7D). These data indicate lysozyme is stable against high RH.


32

Page 33 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


507 508

Figure 7. Effects of humidity on the structure and stability of reconstituted tablet samples of

509

BSA. (A) Percentage of BSA aggregates, analyzed by SEC, at different pressures and RHs

510

relative to uncompressed BSA stored at 4 oC. (B) Rh of BSA by DLS. (C) Thermal unfolding

511

curves of BSA samples monitored by their MSM peak positions of intrinsic fluorescence spectra.

512

Error bars represent standard errors based on data for three samples. (D) DSC thermograms and

513

AUC of the thermograms of BSA (DSC experiments were performed in duplicate).

514 515

DISCUSSION


33


Page 34 of 42

516

The goal of this work was to better understand the material properties of protein and

517

perform a comprehensive analysis of the effects of a variety of compression variables on the

518

structure and stability of protein tablets. Two model proteins (BSA and lysozyme) tested in

519

this study exhibited high hygroscopicity, high plasticity, acceptable tabletability, and RH

520

dependent manufacturability. Investigated variables related to protein stability include

521

particle size, pressure, and RH. Data show that lysozyme is highly resistant to mechanical

522

compression even when it is exposed to highly humid environments, suggesting its high

523

intrinsic stability. In comparison, BSA is labile to compression and protein aggregation was

524

observed for BSA samples exposed to even a low level of pressure (as low as 25 MPa for >500

525

µm). Higher levels of pressure induced more irreversible aggregates in BSA, as evidenced by

526

greater increases in Rh and greater losses of monomer. Such dramatically different responses

527

of BSA and lysozyme to compression suggest that the susceptibility of a protein to

528

compression is highly protein dependent and possibly a function of a protein’s intrinsic

529

stability, surface hydrophobicity, and molecular size.

530

In the case of BSA, it is interesting that changes in protein’s aggregation (association) state

531

were observed while other structural characterization (secondary and tertiary) showed no

532

difference. The largest monomer loss (approximately 5%) was seen for samples with the

533

smallest powder size compressed at the highest pressure (Figure 6D). The absence of such

534

changes in other structural analyses (FTIR, fluorescence and Raman) may indicate that the

535

pressure-induced aggregates had minor or negligible alterations of protein’s secondary and

536

tertiary structures. Possible aggregation pathways of BSA include formation of disulfide


34

Page 35 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


537

bonds, other types of covalent linkage between amino acids, and extremely tightly bound

538

non-covalent aggregation. Further experiments are required to elucidate the aggregation

539

mechanism of BSA samples upon compression.

540

The aggregation level of BSA samples was also found highly dependent on the powder

541

particle size. Compression induces a higher level of aggregation in protein samples with a

542

smaller powder size. The smaller the size of protein powders, the larger the total surface area

543

of the protein. One possibility is that the aggregation primarily occurs among proteins

544

located at the contacting surface upon compression. To test this hypothesis, BSA samples

545

with a series of defined powder particle size ranges can be prepared and compressed at the

546

same level of pressure. A proportionality between increases in aggregation levels and the

547

total particle surface area would confirm this hypothesis. Data collected in this study are not

548

sufficient to establish such a correlation.

549

High RH was also found to accelerate the degradation of BSA, as evidenced by more

550

aggregation at higher RHs for a period of one month at 25 oC. Although compression did

551

further induce aggregation, such increase in aggregation was nearly independent of the RH

552

level at which BSA was incubated. This suggests that RH and compression do not have a

553

synergetic effect on BSA degradation. However, lysozyme was highly resistant to high

554

humidity.

555

dependent. The degradation of BSA was examined immediately after compression. It would

556

also be desirable in the future to investigate the long-term stability of BSA tablets prepared

557

under various RH levels and degradation kinetics of these tablets stored at different

Therefore, the RH effect on a protein’s structure and stability is protein


35


Page 36 of 42

558

temperatures (e.g., 4, 25, 40 ᵒC). In addition, effects of excipients on the structure and

559

stability of protein during compression and over long-term storage would be worth

560

investigation. Excipients that can specifically mask the surface of a protein particle or its

561

local hydration can potentially reduce the aggregation of BSA in tablets, if the hypothesis

562

proposed above is valid.

563 564

CONCLUSION

565

Amorphous BSA and lysozyme are both very hygroscopic. They exhibit lower E and H

566

than most of pharmaceutical organic crystals and biomaterials. Their softness renders

567

tabletability sufficient for making tables without using any excipients. Compression did not

568

lead to detectable changes in secondary and tertiary structures or thermal stability of both

569

proteins after reconstitution. Compression and high humidity also neither affect activity of

570

lysozyme nor cause more aggregation. Changes in environment RH affect properties of the

571

two proteins differently. At RHs higher than 75%, BSA powder caked while lysozyme

572

powder exhibited punch sticking and tablet lamination. Pressure and particle size

573

significantly affected aggregation of BSA. It appears that the development of tablet dosage

574

forms of proteins by compression is possible, provided protein degradation can be prevented

575

by using appropriate formulation and process control strategies.

576 577

ASSOCIATED CONTENT


36

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578

Supporting Information

579

The following file is available free of charge. Raman data, FTIR second derivative spectra,

580

DSC thermograms, SEC chromatograms, DLS, and thermal unfolding curves (PDF)

581

AUTHOR INFORMATION

582

Corresponding Authors

583

*C. Russell Middaugh, Department of Pharmaceutical Chemistry, 2030 Becker Dr.,

584

University of Kansas, Lawrence, KS 66047. E-mail: [email protected]; Telephone: 785-864-

585

5813; Fax: 785-864-5814

586

*Changquan Calvin Sun, Department of Pharmaceutics, University of Minnesota,

587

Minneapolis, MN 55455. E-mail: [email protected]; Tel: 612-624-3722; Fax: 612- 626-2125

588

Present Address

589

∥ Regeneron

590

ORCID

591

Yangjie Wei: 0000-0002-4268-1065

592

Chenguang Wang: 0000-0003-1926-5722

593

Bowen Jiang: 0000-0003-0913-6468

594

Changquan Calvin Sun: 0000-0001-7284-5334

595

Author Contributions

596

⊥

Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA

Y.W. and C.W. contributed equally to this work


37


Page 38 of 42

597

Notes

598

The authors declare no competing financial interest.

599

ACKNOWLEDGMENT

600

This research was supported in part by a Graduate Student Fellowship Award to YW from

601

the American Association of Pharmaceutical Scientists Foundation.


38

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602

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SYNOPSIS The solid state form, mechanical properties, manufacturability, and tabletability, as well as

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the effect of compression on the structure and stability of bovine serum albumin and

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lysozyme were investigated to assess the possibility of developing protein tablets.


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Developing Biologics Tablets: the Effects of Compression on the

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