Trehalose Limits Fragment Antibody Aggregation and Influences

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Trehalose Limits Fragment Antibody Aggregation and Influences Charge Variant Formation in Spray Dried Formulations at Elevated Temperatures Karthikan Rajagopal, Debby Pei-Shan Chang, Purnendu Nayak, Saeed Izadi, Thomas W. Patapoff, Jennifer Zhang, Robert Franklin Kelley, and Alavattam Sreedhara Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01002 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018

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Molecular Pharmaceutics

Trehalose Limits Fragment Antibody Aggregation and Influences Charge Variant Formation in Spray Dried Formulations at Elevated Temperatures Karthikan Rajagopal,1,* Debby Chang,1 Purnendu Nayak,2 Saeed Izadi,3 Thomas Patapoff,3 Jennifer Zhang,4 Robert Kelley1 and Alavattam Sreedhara 5 1Drug

Delivery, Genentech Inc., 1 DNA Way South San Francisco CA 94568

2Eurofins 3Early

Lancaster Laboratories, Lancaster, Pennsylvania PA 17605

Stage Formulation Development, Genentech Inc., 1 DNA Way South San Francisco CA

94568 4Protein

Analytical Chemistry Departments, Genentech Inc., 1 DNA Way South San Francisco

CA 94568 5Late

Stage Formulation Development, Genentech Inc., 1 DNA Way South San Francisco CA

94568 *Corresponding author KEY WORDS solid state, hot-melt extrusion, antibody, trehalose, stability, spray-dried

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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TABLE OF CONTENTS GRAPHIC


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ABSTRACT

The preparation of PLGA rods for sustained release applications via hot-melt extrusion process employs heat and mechanical shear.

Understanding protein stability and degradation

mechanisms at high temperature in solid state is therefore important for the preparation of protein-loaded PLGA rods. The stability of a model protein, labeled Fab2, has been investigated in solid-state formulations containing trehalose at elevated temperatures.

Spray-dried

formulations containing varying levels of trehalose were exposed to temperatures ranging from 90°C to 120°C.

Measurement of aggregation and chemical degradation rates suggests that

trehalose limits Fab2 degradation in a concentration dependent manner but the effect tends to saturate when the mass ratio of trehalose to protein is around one in the solid formulation. The Fab2 secondary structure and spray-dried particle morphology were studied using circular dichroism and scanning electron microscopy techniques respectively. Based on temperature and trehalose-dependent aggregation kinetics as well as changes in spray dried particle morphology a mechanism is proposed for the trehalose stabilization of proteins in solid state at elevated temperatures. The results reported here suggest that when fragment antibodies in solid state are formulated with trehalose as excipient a high temperature process such as hot-melt extrusion can be successfully accomplished with minimal degradation.


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INTRODUCTION Protein-based therapeutics such as monoclonal (Mab) and fragment (Fab) antibodies has garnered immense interest in the last couple of decades.1-4 Antibodies are a sub-class of proteins that have become an important part of the therapeutic arsenal for several diseases including treatments in oncology, immunology and opthalmology. The treatment of many chronic diseases necessitates continuous infusions and/or periodic injections but such dosing regimens are associated with severe patient compliance issues.5-8 Sustained drug delivery approaches from polymer-based systems and devices can overcome patient compliance challenges and also provide convenience during administration.

However, maintaining drug stability during

processing, long-term storage and in vivo delivery from sustained release formulations is a major challenge particularly for protein-based drugs. Consequently, there are several sustained release technologies in market for the delivery of small molecules and peptides but none for proteins. The preparation of polymer-based drug delivery systems such as solid rods (or implants)9, microspheres10 and in-situ forming depots11 utilize processing conditions that can potentially impact drug stability. Heat and mechanical shear, for example, are employed during the preparation of polymer rods.9,

12-13

The physical and chemical ‘fragility’ of protein-based

therapeutics may impose limitations on the processing conditions that could be employed for the preparation of such polymeric systems. As such, the nature of stresses employed and their impact on protein stability may dictate the choice of delivery technology for that therapeutic modality. For example, drug instability at elevated temperatures may prevent the use of a hotmelt extrusion process for the preparation of polymer rods. An understanding of protein stability and degradation pathways under certain processing conditions may enable the design of robust formulations for the development of new drug delivery technologies.14-15


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A solid drug formulation such as spray-dried powder is preferred for the preparation of many polymer-based sustained release systems.9, 11 It was recently demonstrated that proteins in spray-dried form enable long-term release from solid polymer implants9 and polymer-solvent depots11. In contrast to lyophilization, spray-drying directly produces micron-sized particles that are readily amenable to solid state processing with hydrophobic polymers such as poly(lactideco-lactide) (PLGA). In addition, solid protein particles are generally inert in non-polar and aprotic solvents (unpublished data). Antibodies in solid formulations generally exhibit increased resistance to degradation when subjected to heat stress.16-17 Slower degradation rates in solid-state formulations stems from the suppression of both the slow and long-range molecular mobility (-relaxations), and the fast and local structural relaxations (-relaxations), and the suppression of water-mediated chemical degradation reactions in amorphous formulations (below glass transition temperature, Tg).17 It is therefore not surprising to note that the denaturation or unfolding temperature (Tm) of a protein in a solid formulation is far higher than in aqueous formulation and is usually above the Tg of the solid formulation.18 Nevertheless, aggregation and side-chain chemical modifications potentially leading to a loss in product quality can occur at temperatures well below the formulation Tg provided the exposure duration is sufficiently long. Thus, a combination of temperature, formulation Tg and the exposure duration govern protein stability at high temperatures in solid state. Fragment antibody (Fab) stability in solid formulations at elevated temperatures is of particular interest here. In the preparation of polymer implants via a hot-melt extrusion process the drug is exposed to elevated temperatures - typically in the range 70C to 110C - for about 30 minutes.

An understanding of antibody degradation pathways and their rates at elevated


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temperatures is essential for establishing both the maximum operation temperature and the exposure duration during processing. Moreover, knowledge of antibody degradation in solid formulations at elevated temperatures could prove useful in excipient selection for maintaining stability during processing. Sugars such as trehalose and sucrose are generally known to protect antibodies in solid formulations but systematic studies for understanding the effect of sugar at elevated temperatures as described here have not been reported before. It is based on the presumption that physical and chemical degradation will be inevitable at elevated temperatures but careful formulation design can minimize degradation such that there is minimal or no loss to product quality. In this report, we have assessed the physical and chemical stability of Fab2, a 47 kD fragment antibody, in spray-dried formulations after heat-treatment at elevated temperatures. A temperature range between 90C and 120C was chosen for investigation to assess if proteins can be processed at elevated temperatures for the preparation of polymer-based formulations prepared by hot-melt extrusion process. The second objective of the study was to decipher the impact of trehalose on Fab2 stability in solid state and the spray-dried particle morphology. Specifically, we have probed the aggregation behavior, chemical degradation and secondary structure as a function of temperature and trehalose concentration in spray-dried formulations. Finally, based on the stability data, changes in spray-dried particle morphology and simple model calculations we have proposed a mechanism that can account for the temperaturedependent changes in solid-state protein stability. MATERIALS AND METHODS


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1. Materials: Fab2 was obtained from Genentech (South San Francisco, CA). α-α trehalose dihydrate was obtained from Ferro Pfanstiehl Laboratories (Cleveland, OH). Histidine-HCl was obtained from Sigma-Aldrich (St. Louis, MO) and polysorbate 20 (PS20) was obtained from Spectrum Chemical (New Burnswick, NJ) 2. Spray drying:

Fab2 at 25 mg/mL was dialyzed (MWCO 10000 Da) against 10 mM

histidine/histidine-HCl buffer (pH 5.5) containing 0.01% polysorbate 20.

After overnight

dialysis and changing the dialysis buffer four times Fab2 was diluted to 10 mg/mL with 10 mM histidine/histidine-HCl buffer (pH 5.5) containing 0.01% polysorbate 20. UV spectroscopy was used to measure Fab2 concentration (=1.39 mL.cm-1.mg-1 at 280 nm). The required amount of trehalose dihydrate was added to Fab2 solution as per Table 1 and the aqueous formulations were spray-dried using Buchi Model 191 laboratory-scale spray drier (New Castle, NJ) at an inlet and outlet temperature of 89+2 °C and 59+2 °C respectively. The pump power was 10% and the aspirator was operated at 100% capacity. The liquid feed rate was 3.4 mL/minute and the compressed air flow rate was 600 L/hr. The collected spray-dried powders were transferred to a clean, dry glass vial and stored under nitrogen in a vacuum chamber until further use. 3. Scanning electron microscopy (SEM): The morphology of the spray-dried particles was imaged with a Quanta 3D (Hillsboro, OR) FEG SEM. The samples were fixed on aluminum stubs with carbon adhesive tape and sputter coated with gold/palladium (Cressington Sputter Coater, TED Pella, Inc.) to improve their electrical conductivity. SEM images were collected at low voltage to minimize any potential sample damage or surface charging. 3. High temperature heat treatment: Two to seven milligram of spray-dried powder (equivalent to 1 to 1.5 mg Fab mass) from each formulation was weighed into a 7 mL glass vial.


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The vials were uncapped and placed in a Binder forced-air convection oven (Bohemia, NY) preheated and equilibrated to the desired temperature between 90 and 120C. Samples were removed at 1 hr, 3 hrs, and 5 hrs, capped immediately and allowed to cool to room temperature. The vial contents were dissolved in purified water such that the final Fab2 concentration was ~ 1 mg/mL. The reconstituted aqueous samples were observed for clarity and visibly clear samples were used for SEC and IEC analysis. 4. Size-exclusion chromatography (SEC): Size-exclusion chromatography (SEC) HPLC was performed using an Agilent 1200 series HPLC system (Santa Clara, CA) equipped with a TOSOH TSKgel G2000SWXL (30 cm x 7.8 mm i.d., 5 µm particle size) column. Samples were analyzed at 25 °C in isocratic mode with 0.20 M K3PO4, 0.25 M KCl, pH 6.2 as mobile phase at a flow rate of 0.7 ml/min. A 20L sample at 1 mg/mL concentration was injected and the total run time was 20 min. 5. Ion-exchange chromatography (IEC): IEC was performed using an Agilent 1200 series HPLC system (Santa Clara, CA) on two Dionex (Sunnyvale, CA) ProPac® SAX-10 (2 mm × 250 mm) strong anion-exchange columns connected in series and equipped with a diode array detector (DAD). Mobile phase A (solvent A) was 20 mM Tris buffer, pH 8.2) and mobile phase B (solvent B) was 250 mM sodium chloride dissolved in solvent A. Prior to analysis, the samples were diluted to approximately 1mg/mL in solvent A and 20L sample was injected. A linear gradient starting from 100% solvent A at 0 minutes to 20% solvent A at 45 minutes was employed to separate Fab2 charge variants in a total of ~60 min run time. Absorbance at 280 nm was used to detection. The IEC peaks were separated into main peak, acidic peak, and basic peak.


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6. Circular dichroism: Near and far-UV CD spectra were collected using a JASCO J-815 CD spectrophotometer (Easton, MD). Spray-dried Fab2 samples were dissolved in purified water to get an approximate concentration of 0.3mg/mL. Aqueous samples were prepared in pH 5.5 buffer (10 mM histidine-HCl/histidine). Precise Fab2 concentration before CD spectroscopy was determined using UV absorption at 280 nm using a molar extinction coefficient of 43,824 M1cm-1.

Wavelength spectra were collected at 25C from 190 nm to 250 nm at 1 nm interval in a

0.1 cm quartz cuvette. Mean residue ellipticity [θ] (deg cm2/dmol) was calculated from the equation [θ] = (θobs/10lc)/r, where θobs is the measured ellipticity in millidegrees, l is the pathlength of the cuvette (cm), c is the molar concentration, and r is the number of amino-acid residues (r = 463 for Fab2). 7. Estimation of trehalose binding sites on Fab2: Molecular Operating Environment (MOE) software was used for constructing the homology model of the Fab from its sequence. To estimate the surface areas of trehalose molecule and the Fab surface accessible to binding, the solvent excluded surface (SES) area for one face of a single trehalose molecule (half of the total SES) and the total solvent accessible surface area (SAS) of the Fab was calculated using the AREAIMOL software.19 The SAS value was used for the Fab in an attempt to minimize the effects of “isolated” surface areas of the Fab, which could be cavities either within the molecule or formed as a result of intermolecular contacts. However, the trehalose molecule is small with no buried surfaces, therefore the SES value should be more realistic for estimating the surface accessible to binding. By calculating the ratio of Fab SAS area (~20150 Å2) to the half of the trehalose SES area (~154Å2), the total number of sugar molecules that can theoretically bind to the Fab surface is obtained as ~130.


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8. Differential scanning calorimetry (DSC): DSC was performed on TA Q200 (New Castle DE) under modulation condition. Approximately 2+1 mg sample was weighed in an aluminium pan and hermetically sealed. The sample was equilibrated at 5°C for 10 minutes and then heated at 2°C/min with +/- 1.00°C/min modulation to 120°C and cooled back to 5°C at 2°C/min. After equilibrating the sample again at 5°C the same heating and modulation ramp was repeated for a second time all the way to 180°C. The first heating ramp was done to eliminate any thermal history associated with the sample because of storage or handling conditions. For reporting purposes the second heating ramp of sample was used which is free from any thermal history. RESULTS 1. Characteristics of spray-dried Fab2 formulations Table 1 lists the details of six spray-dried Fab2 formulations. Depending on the trehalose amount in the formulation, the total solid content in the aqueous formulation before spray drying was between 12 mg/mL and 112 mg/mL. Accordingly, the formulations were parameterized using trehalose to protein ratio (T/P). For the formulations listed in Table 1, T/P ratio was between 0 to 10 on mass basis or 0 and 1240 on molar basis. All formulations after spray drying appeared white and were dry powders. The physical and chemical stability of Fab2 after spray drying was assessed using ionexchange (IEC) and size-exclusion chromatography (SEC) respectively (see Table 1). Before spray drying, the monomer content (SEC) and main peak (IEC) in Fab2 was 99.5% and 89% respectively. Visibly clear solutions were obtained upon reconstitution of all the spray-dried powders. In the formulation devoid of trehalose approximately 0.3% monomer was lost to aggregation during the spray-drying process. In all the formulations containing trehalose no


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appreciable change was observed in the charge variants (main peak by IEC) or the monomer content (by SEC) due to spray-drying. Table 1: Fab2 formulations before spray drying and stability after spray drying Formulation

Antibody (mg/mL)

Trehalose (mg/mL)

Trehalose/ Protein (T/P) (mg/mg)

Trehalose/ Protein (T/P) (mol/mol)

Total Solids (mg/mL)

% Monomer (SEC)

% Main Peak (IEC)

A

10

0

0

0

12

99.0

85.4

B

10

3.3

0.33

41

15.3

99.5

87.2

C

10

6.6

0.66

82

18.6

99.5

88.1

D

10

10

1

124

22

99.6

89.3

E

10

50

5

620

62

99.6

90.1

F

10

100

10

1240

112

99.6

89.6

Figure 1 shows the representative SEM micrograph of spray-dried Fab2 particles prepared from the formulation with T/P ratios 0, 1.0 and 10.0. At T/P ratio of 0 and 1.0 the spray-dried particles exhibit a dimpled surface morphology. At T/P ratio of 10.0 the particles appear spherical with a smooth surface morphology. The spray-dried particle size is a few microns and does not change with sugar content.


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A

B

C

Figure 1. Morphology of spray dried Fab2: SEM micrograph of spray dried Fab2 from formulation with T/P ratio 0 (A), 1.0 (B) and 10.0 (C). Scale bar is 2 m.


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2. Aggregation in spray dried formulations The stability of Fab2 in spray-dried formulations was studied at temperatures ranging from 90°C to 120°C. Figure 2 shows the change in monomer fraction (f) with time (t) for all six spray-dried formulations after heat treatment at 90°C, 100°C, 110°C and 120°C for a few hours. The decrease in monomer content was due to the formation of dimers and trimers (soluble higher molecular weight aggregates). At 110°C (Figure 2C) a clear trend indicating an increase in aggregation rate with decreasing trehalose content can be observed.

Similar trends were

maintained at 90°C and 100°C, albeit at slower aggregation rates. At 120°C (Figure 2D), however, a rapid loss in monomer content due to heat treatment was observed. Importantly, the monomer loss at 120°C does not correlate with the trehalose content in the formulation. The exposure duration at elevated temperatures was limited to five hours as in some cases longer exposure time led to the formation of insoluble precipitates upon reconstitution. Assuming antibody aggregation within solid formulations to be a first-order process, the aggregation rate constant k was estimated from the slopes of linear fits to ln (f) vs t data. Interestingly, at T < 110C the rate constants (k) increased exponentially with temperature (T), thus suggesting that Fab aggregation approximates to Arrhenius kinetics.

Consequently,

reasonable fits for ln (k) vs 1/T data were obtained (Figure 3A). Formulations containing higher trehalose (T/P > 5.0) could not be included in this analysis as aggregation was barely detectable by SEC for quantification at temperatures < 100°C. To understand the impact of trehalose content on antibody aggregation, the rate constant k was plotted as a function of T/P ratio for temperatures up to 110C. Increasing the trehalose content (T/P ratio) in the formulation decreases k but the effect is clearly non-linear (Figure 3B).


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A dramatic decrease in aggregation rate was observed at high T/P ratios but the trehalose effect on aggregation tends to saturate above T/P = 1.0 on mass basis or 124 on mole basis.

A

B

90°C

100°C 1

Monomer Fraction, f

Monomer Fraction, f

1

0.9

T/P = 0 T/P = 0.33 T/P = 0.66 T/P = 1 T/P = 5.0 T/P = 10

0.8

0.7

0

1

2

3

4

Time, t (hours)

C

5

T/P = 0 T/P = 0.33 T/P = 0.66 T/P = 1.0 T/P = 5.0 T/P = 10.0

0.8

0

1

2

3

4

Time, t (hours)

D

110°C

5

6

120°C 1

Monomer Fraction, f

0.9

T/P = 0 T/P = 0.33 T/P = 0.66 T/P = 1.0 T/P = 5.0 T/P = 10.0

0.8

0.7

0.9

0.7

6

1

Monomer Fraction, f

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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0

1

2

3

4

Time, t (hours)

5

6

0.9

T/P = 0 T/P = 0.33 T/P = 0.66 T/P = 1.0 T/P = 5.0 T/P = 10

0.8

0.7

0

1

2

3

4

Time, t (hours)

5

6

Figure 2. Fab2 aggregation in spray dried formulations: The change in monomer fraction (f) as a function of time (t) for Fab2 in spray dried formulations after exposure to 90°C (A), 100°C (B), 110°C (C) and 120°C (D) is shown. The lines in all panels are linear fits to f vs t data.


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Arrows in plots A, B and C indicate the change in trend as a result of increasing trehalose concentration (T/P mass ratio) in the formulation.

B

-2 T/P = 0 T/P = 0.33 T/P = 0.66 T/P = 1.0

-3

Rate Constant, k (hr-1)

A Rate Constant, k (hr-1)

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


-4 -5 -6 -7 2.6

0.1

0.08

1/T

2.7

2.75

T/P = 1 (mg/mg) or 124 (mole/mole)

0.06

0.04

0.02

ln (k) = ln (A) – (Ea/R) (1/T)

2.65

90C 100C 110C

2.8

0

0

(x10-3, °C)

400

800

T/P (mole/mole)

1200

Figure 3. Analysis of Fab2 Aggregation: A First-order rate constant (k) is plotted as a function of inverse temperature (1/T) for four T/P formulations. Temperature dependent aggregation follows Arrhenius kinetics and is fit to ln(k) = ln(A) – (Ea/R) (1/T), where A is a pre-exponential factor, Ea is the activation energy, R is gas constant. R2 for all fits ≥ 0.99. B Change in rate constant (k) with trehalose concentration (T/P) is shown at 90°C, 100°C and 110°C. 3. Chemical degradation in spray dried formulations IEC was used to analyze chemical degradation in Fab2.

Figure 4A shows the

representative IEC of Fab2 sample that was generated after exposing an aqueous formulation in pH 5.5 buffer to 40C for four weeks. The IEC method employed a strong anion exchange column for charge-based separation.

Two prominent chemical degradation modes can be

identified in Fab2: (1) cyclization of heavy chain N-terminal glutamic acid (Figure 4B) leading


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to pyroglutamate (pE) formation, and (2) cyclization of aspartic acid side chains in the heavy (HC) and light chains (LC) of the Fab leading to


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A Basic Peaks

96

11

Asu

Main Peak

80

6

Absorbance

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


60

+pE + Asu +pE

+pE 3

40

Acidic Peaks

7

20

8

5 1 20.0

17.6

10

4

2

12

9

22.5

25.0

27.5

30.0

32.5

35.0

37.5

40.0

42.2

Minutes

B O

-H2O

HO

O

N H

H2N

O

O

N-terminal Glu

Pyroglutamate (pE)

C

O OH N H O

O OH

N H

H N O

Asp

-H2O

+H2O

H N O

Asp

N

N H

O

O

Asu

+H2O

HN OH

N H O

Iso-Asp Figure 4. Chemical degradation modes of Fab2: A Ion-exchange chromatogram of chemically degraded Fab2. Peaks corresponding to N-terminal pyroglutamate formation are identified as


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pE. B Pyroglutamate formation via cyclization of N-terminal glutamic acid. C Succininde intermediate (Asu) formation from aspartic acid side chain and its subsequent hydrolysis leading to iso-aspartic acid formation (Iso-Asp).

succinimide intermediate product (Asu) and isoaspartate (Iso-Asp) formation (Figure 4C). Since pyroglutamate formation and aspartic acid cyclization degradation modes eliminate a negative charge each on the molecule, charge variants are generally observed as basic peaks on anionic IEC. The main peak (peak 11) and the individual basic peaks including peaks 10, 6 and 5 (in Figure 4A) was isolated and analyzed using tryptic peptide mapping by LC-MS (data not shown). Peak 10 corresponds to the N-terminal pyroglutamate version of an otherwise undegraded Fab2 molecule. Peaks 6, 7 and 8 correspond to the succinimide intermediate product on LC-Asp30, LC-Asp31 and HA-Asp32 respectively.

Peak 5 contains both the succinimide

intermediate product on LC-Asp30 and the N-terminal pyroglutamate. For analysis purpose the ratio of the height of peaks 10 and 11 was used as a measure of pyroglutamate formation. It is important to note that the stress employed for generating the charge variants in Figure 4A was pH 5.5 and 40°C. Under this pH condition, acidic peaks corresponding to deamidation of asparagine side chains are usually not observed at a significant level. Figure 5A shows the IEC of Fab2 in T/P = 1 formulation after exposure to 100C. Clearly, increasing the exposure duration causes a progressive increase in peaks corresponding to pyroglutamate (peaks 5 and 10) and succinimide product (peak 6). Figure 5B shows the IEC for all T/P formulations after exposure to 100°C for 3 hours. Distinct trehalose concentrationdependent changes in peak contours can be noted – i.e. increasing the trehalose concentration


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B

T/P = 1 Formulation at 100°C

0 hr 1 hr 3 hr 5 hr 6 10 5

26

28

30

32

After 3hrs at 100°C

Relative Absorbance

Relative Absorbance

A

34

36

C

5

26

28

Ratio (Peak10/Peak11)

90C 100C 110C 120C

10

30

32

Time (min)

32

34

36

D 0.8

T/P = 1 Formulation After 3 hrs

28

30

Time (min)

5

26

T/P = 0 10 T/P = 0.33 T/P = 0.66 T/P = 1.0 T/P = 5.0 T/P = 10.0

6

Time (min)

Relative Absorbance

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


34

36

T/P = 0 T/P = 0.33 T/P = 0.66 T/P = 1.0 T/P = 5.0 T/P = 10.0

0.6

0.4

0.2

0

0

1

2

3

4

Time, t (hours)

5

6

Figure 5. Chemical degradation of Fab2 in spray dried formulations at high temperatures: A IEC of Fab2 in T/P = 1.0 formulation as a function of time after exposure to 100°C. B IEC of all Fab2 formulations after three-hour exposure to 100°C.

C IEC of Fab2 in T/P = 1.0

formulation after 3-hour exposure to temperatures ranging from 90°C to 120°C. Arrows in all plots indicate the changing trend in peak contours due to increase in exposure time (A) or increase in trehalose concentration (B) or increase in temperature (C).

All ion-exchange

chromatograms have been normalized to main peak height to emphasize the relative change in


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degradation products. D Change in ratio of peak 10 to peak 11 (see Fig 4) with time at 90°C for all formulations. The change peak ratio is a measure of extent of pyroglutamate formation. The arrow indicates reduction in pyroglutamate formation with increasing trehalose in Fab2 formulations. causes a decrease in pyroglutamate formation (peaks 10) and an increase in succinimide product (Asu, peak 6). The suppression of pyroglutamate formation by trehalose can also be noted in the decrease of peak 5, which is the pyroglutamate version of the succinimide product (peak 6). Figure 5C shows the IEC profile in T/P =1 formulation after 3 hours at various temperatures. Increasing the exposure temperature causes an increase in chemical degradation but the effect becomes dramatic at 120°C. After 3 hours at 120C nearly all of the N-terminal glutamic acid (peaks 5 and 10) and most of the LC Asp 30 has been converted to the respective cyclic intermediate product. 4. Temperature dependent changes in Fab2 conformation Fab2 conformation after heat treatment was assessed using circular dichroism (CD) spectroscopy. For comparison, Fab2 conformation in aqueous formulation was analyzed after heating to 85°C for 1 hour. Fab2 conformation in spray-dried form after heat treatment at 100°C and 135°C was analyzed after cooling and reconstitution in water. Far-UV CD spectra (Figure 6A) clearly suggests that Fab2, when heated to 85°C in aqueous formulation, undergoes irreversible aggregation. The increase in CD signal around 215 nm in all three formulations after heat treatment suggests inter-molecular aggregation via -sheet formation. Importantly, the presence of trehalose in the aqueous formulation does not seem to have any impact on the secondary structure. In solid state, however, far-UV CD spectroscopy (Figure 6B) could not detect any changes in the secondary structure after exposure to 100°C for 1 hour even in the


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formulation devoid of trehalose. Slight changes in the near-UV spectra can however be noted. Spray-dried samples heated to 135°C could not be analyzed by CD spectroscopy as they formed a cloudy solution upon reconstitution.

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Figure 6. Conformational stability of Fab2: A Far-UV and near-UV CD spectra of Fab2 in aqueous formulation buffer before and after heating to 85°C for 1 hour. B Far-UV and near-UV CD spectra of Fab2 in spray dried formulation before and after exposure to 100°C for 1 hr. The spray-dried sample was re-constituted in water before measurement of spectrum. 5. Temperature dependent changes in particle morphology DSC could not unequivocally establish Tg for T/P=0 and T/P=1.0 formulations (Figure S1). For T/P = 10 formulation, glass transition could be noted at 111°C. It was of interest to see


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if any changes in particle morphology can be used to infer mobility of formulation components. Particle morphology was assessed using scanning electron microscopy (SEM). Figure 7 shows the SEM images of spray dried powders before and after heat treatment at 100°C, 135°C and 170°C for the three T/P formulations. Particle morphology (Figure 7, top row panels) before heat treatment depends on the trehalose content in the formulation. In T/P = 0 and T/P = 1 formulations, large surface dimples in otherwise spherical particles were observed. Whereas in the T/P = 10 formulation, the particles were completely spherical without any dimples. After exposure to 100°C for one hour, SEM detected no change in particle morphology for all three formulations. At 135°C, distinct morphological change in T/P = 10 formulation could be observed. The spherical particles in T/P = 10 formulation at T < 100°C appear to have melted and fused to form a single homogeneous macroscopic surface. At 170°C the ‘melting and fusion effect’ appears to have occurred in T/P = 1 formulation as well.

Interestingly, T/P = 0 formulation showed no change in particle

morphology even after onehour heating at 170°C. Visually however the changes in particle morphology due to heat treatment were not perceptible. DISCUSSION Improved protein stability in solid state is generally accounted using two hypotheses – the water replacement (thermodynamic) and the glassy state (kinetic) hypothesis.

16, 20-23

The

water-replacement hypothesis posits that in solid state the sugar molecules replace the water molecules bound to the protein surface in aqueous phase. The thermodynamically stabilizing protein-water interactions in aqueous phase are replaced by protein-sugar interactions in solid


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T/P = 1

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2 µm

Figure 7. Morphology of spray dried particles: SEM images of Fab2 spray dried particles before and after heat treatment for 1 hour at various temperatures. Note the change in particle morphology in trehalose containing formulations after heating to 170°C for 1 hour.


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state. Alternatively, the glassy state hypothesis suggests that the protein molecules are immobilized within a rigid and amorphous (glassy) sugar matrix. In this case the sugar matrix retards chemical degradation reactions and also maintains separation between neighboring protein molecules. Potentially, both of the aforementioned mechanisms could simultaneously contribute to improved stability in solid state. Moreover, even if large-scale molecular mobility (-relaxations) is attenuated in solid-state formulations, local structural relaxations, primarily involving rotation around single bonds (-relaxations), and intermolecular association of Fab molecules adjacent to each other may still persist and contribute to protein chemical degradation and aggregation in solid state. The data presented here confirms that trehalose suppresses Fab aggregation in solid-state formulations even at elevated temperatures. The seeming saturation in aggregation rates at T/P > 1 implies that trehalose ‘coats’ the protein surface. The ‘coating’ process presumably occurs during the spray-drying process. After all the trehalose-binding sites on the protein surface have been occupied the excess trehalose exists in a separate amorphous phase. Trehalose in excess of 125 molecules per Fab molecule is not expected to contribute to stability improvement. Trehalose binding is presumably non-specific and could involve hydrogen-bonding interactions between sugar molecules and the protein amide bonds and hydrophilic amino acid side chains. Interestingly, in a similar but long-term study performed at 37°C, Andya et al. found that 375 trehalose molecules are required for the maximal stability of a Mab molecule in solid state.24 The MW and surface area of a Mab is approximately three times that of a Fab and experiments were conducted at a much lower temperature but the findings are consistent – maximal excipientdependent stability is achieved when the sugar and protein masses in the formulation are nearly equal – i.e. T/P = 1 by mass.


24

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An indirect but convincing verification for trehalose ‘coating’ hypothesis can be obtained from the estimation of the number of sugar molecules that can theoretically bind to antibody

Figure 8. Distribution of Fab2 in spray dried particles: A Solvent accessible surface area of Fab2 (blue) and solvent excluded surface area of trehalose (yellow). The distribution of trehalose around Fab2 is shown to demonstrate the relative sizes of Fab2 and trehalose. B Three possible modes for Fab2 distribution within micron-sized spray dried particles is presented.


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surface based on the solvent accessible surface area of the Fab and the solvent excluded surface area of one face of a single trehalose molecule. As per this calculation (Figure 8A), 130 trehalose molecules will be needed to completely cover the surface of Fab2. Interestingly, this is approximately equivalent to the trehalose content (125 trehalose molecules per Fab) at which saturation in aggregation kinetics is observed in the current study. Three types of intermolecular interactions can be envisioned in solid protein-sugar formulations: 1. protein-protein, 2. protein-sugar and 3. sugar-sugar interactions.

Protein

stability, however, is dependent on the nature of all interactions involving the protein. Pikal et al. recently demonstrated that sugar-protein mixing at molecular level is necessary for improved protein stability in solid state.25 In a sugar-free formulation (T/P = 0) intermolecular aggregation is simply governed by protein-protein interactions in solid state. In T/P = 1 formulation, if all of the sugar is bound to the protein surface, then the nature of protein-sugar interactions can be expected to govern protein stability. In formulations containing excess sugar (T/P > 1), sugarsugar interactions are present but these interactions are not expected to have any direct bearing on protein stability. The data presented in Figure 3B affirms this hypothesis. In high sugar solid formulations three possible scenarios can be envisioned for sugarantibody distributions (Figure 8B). In the first scenario, the antibody is evenly distributed within a rigid sugar matrix. This would imply that the sugar molecules occupy the intervening space between randomly distributed Fab molecules. Consequently, the average inter Fab separation distance should progressively increase with increasing fraction of sugar in the formulation. For example, the average separation distance between any two Fab molecules in T/P = 10 formulation should be greater than in T/P = 1 formulation. Trehalose concentration-dependent aggregation data reported here however does not support this possibility. A monotonic decrease


26

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in aggregation rate with increase in trehalose fraction would be expected instead of a suppression in aggregation kinetics at T/P > 1.0 (Figure 3B). The fraction of protein is ten-fold higher for T/P = 1.0 formulation compared to T/P = 10 formulation, but the difference in aggregation rate constants (k) is indistinguishable.

The aggregation rate at 110°C is small but is clearly

measurable, and importantly, remains very low in T/P > 1.0 formulations.

In the second

scenario, the protein and sugar exist in separate phases. This is also an unlikely scenario because the sugar clearly impacts protein stability in T/P 1.0) formulations. Given that the aggregation rates plateau and that there is no appreciable difference in aggregation rates at T/P > 1.0, enrichment of antibody molecules within the solid formulation seems a likely scenario. In a related study, Grasmeir et al. predicted that proteins preferentially accumulate at the droplet surface.26 The results reported here does not preclude this possibility. It is likely that proteins accumulate at the interface along with a layer of sugar particularly because in solid state the sugar molecules can replace water molecules; i.e. protein enrichment at the interface is a possibility. It is unique and interesting to note that while trehalose suppresses pyroglutamate formation it increases the succininde intermediate product (Figure 5). Pyroglutamation is the cyclization of the N-terminal glutamic acid. Hydrogen bonding interaction between N-terminal glutamic acid side chain and the trehalose hydroxyl groups in solid state could possibly explain this observation but it is unclear how trehalose causes an increase in the succinimide intermediate product.

Interestingly, the dramatic change in aggregation and chemical

degradation rates and the lack of trehalose concentration dependence on stability at 120°C


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suggests a possible change in the nature of trehalose-protein interaction between 110°C and 120°C. Further investigation is required for understanding the nature of these interactions and the concomitant temperature-dependent changes. The temperature-dependent changes in particle morphology to some extent reveal largescale translational mobility of formulation components and possibly the nature of intermolecular interactions.

The three different formulations and temperatures (Figure 7) were carefully

selected to see if temperature-dependent changes in particle morphology can be attributed to the mobility of any of the components in the formulation. In the sugar-free formulation (T/P = 0), the surface morphology of the spray-dried particle does not change even after heating to 170°C for one hour. Nevertheless, maximum Fab aggregation is observed in this formulation. The higher aggregation levels in an apparently rigid matrix suggests that inter-molecular association of proteins next to each other presumably drives the aggregation process even though large-scale protein mobility is suppressed. In the T/P = 1 formulation a change in surface morphology at 170°C is clearly suggestive of the mobility of protein-bound trehalose. Interestingly, SEM does not reveal any large-scale trehalose mobility at T ≤ 135°C. In the T/P = 10 formulation, the fusion of the spray-dried particles at T > 135°C is suggestive of the mobility of excess, unbound and amorphous trehalose. The Tg of pure trehalose in amorphous form is 110°C. If the excess trehalose in T/P > 1.0 formulation is in amorphous form then at 135°C the samples are clearly above the Tg of trehalose. For multicomponent and heterogeneous solid-state compositions such as spray-dried biopharmaceuticals a singular Tg cannot be established.

The physical state and hence the

chemical potential of sugar molecules interacting with the antibody surface is different from the sugar molecules not directly interacting with the antibody. Consequently, more than one thermal


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transition related to trehalose mobility can be envisioned. Some of these transitions however may not be calorimetrically measurable as the endothermic heat change is small. The residual water present in a solid formulation can mediate antibody degradation pathways such as inter-molecular aggregation and side-chain chemical modifications.

The

residual water (mostly unbound water) can acts as a plasticizer and effectively reduces the formulation Tg. Increased molecular mobility above Tg can facilitate aggregation and chemical degradation reactions. Alternatively, the water molecule can also initiate hydrolytic degradation reactions such as deamidation, isomerization and main chain fragmentation reactions. Herein, the experiments were performed at temperatures above 90°C. Due to its relatively high vapor pressure at 90°C, most of the unbound water is likely evaporated from the solid formulation. The initial moisture content and the relative humidity of the environment therefore are not expected to greatly impact protein stability at T > 90°C. This was confirmed experimentally by measuring Fab2 stability at 90°C in spray-dried formulations containing less than 1% and 8% initial moisture content (Figure S2).

Lyophilization (bench-top lyophilizer) was used as a

secondary drying process to reduce the initial moisture content in spray-dried samples. The rate of change in aggregates and main peak loss was similar for samples containing high and low initial moisture content. This confirms that the initial moisture content in the spray-dried samples does not impact the stability profile at T > 90°C. That trehalose can provide protection to Fabs up to 110°C in solid state is a key finding in this study.

Trehalose-containing protein formulations are therefore amenable to high

temperature processing methods such as hot-melt extrusion.

The results demonstrate that

trehalose content, processing temperature, exposure duration and a protein’s inherent propensity


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for aggregation and chemical degradation can be balanced to design a formulation such that high temperature processing can be accomplished with minimal protein degradation. CONCLUSIONS The stability of a fragment antibody (Fab) in spray-dried formulations was investigated as a function of trehalose concentration and temperature. Measurement of Fab aggregation in solid state at elevated temperatures suggests that trehalose limits its aggregation but the trehalose effect tends to saturate when the trehalose to Fab ratio is 1 on mass basis. Based on simple surface area calculations this is the ratio at which trehalose effectively covers the protein surface. Interestingly, the trehalose-dependent stabilization of Fab2 was completely lost at 120°C. In addition, trehalose also suppresses pyroglutamation of the N-terminal glutamic acid, however, it has the opposite effect on succinimide product formation in aspartic acid. Based on the trehalose and temperature dependent changes in Fab aggregation kinetics it appears that proteins in spraydried formulations containing trehalose exist within enriched regions. In summary, trehalose stabilizes proteins in solid state at elevated temperatures such that hot-melt extrusion can be successfully utilized for the preparation of protein-loaded polymer implants. ACKNOWLEDGEMENT KR thanks Ben Walters, Li Yi and John Wang for reviewing the manuscript. SUPPORTING INFORMATION Figures S1 & S2


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Trehalose Limits Fragment Antibody Aggregation and Influences

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