Peptide Dendrons as Thermal-Stability Amplifiers for Immunoglobulin

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Peptide dendrons as thermal stability amplifiers for IgG1 monoclonal antibody biotherapeutics Rohit Bansal, Sameer Dhawan, Soumili Chatterjee, Govind Maurya, V. Haridas, and Anurag Singh Rathore Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00389 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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Bioconjugate Chemistry

Peptide dendrons as thermal stability amplifiers for IgG1 monoclonal antibody biotherapeutics Rohit Bansal,b Sameer Dhawan,a Soumili Chattopadhyay,b Govind P. Maurya,a V. Haridasa, and Anurag S. Rathore*b . a Department of Chemistry, Indian Institute of Technology Delhi b Department of Chemical Engineering, Indian Institute of Technology Delhi

*Corresponding author at: Anurag S. Rathore, Ph.D. Professor, Department of Chemical Engineering Coordinator, DBT Center of Excellence for Biopharmaceutical Technology Indian Institute of Technology, Delhi Hauz Khas, New Delhi, 110016, India Phone: +91-11-26591098 Mobile: +91-9650770650 Fax: +91-1126581120 Email: [email protected] Website: www.biotechCMZ.com Abstract 1 ACS Paragon Plus Environment


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Biotherapeutics such as monoclonal antibodies (mAbs) have a major share of the pharmaceutical industry for treatment of life-threatening chronic diseases such as cancer, skin ailments and immune disorders. Instabilities such as aggregation, fragmentation, oxidation and reduction have resulted in the practice of storing these products at low temperatures (-80ºC to -20ºC). However, reliable storage at these temperatures can be a challenge, particularly in developing and underdeveloped countries and hence lately there has been a renewed interest in creating formulations that would offer stability at higher temperatures (25ºC to 55ºC). Most therapeutic formulations contain excipients like salts, sugars, amino acids, surfactants, and polymers to provide stability to the biotherapeutic but their efficacy at high temperatures is limited. The current work proposes use of peptide dendrons of different generations to create formulations that would be stable at high temperature. Amongst these dendrons, third generation lysine dendron L6 has been identified to provide highest stability to mAbs as demonstrated by a host of analytical techniques such as Size Exclusion Chromatography (SEC), Dynamic Light Scattering (DLS), and Circular Dichroism (CD). The bio-compatibility of these dendrons was confirmed by haemolytic activity tests. Non-interference of the dendrons with the activity of the mAb was confirmed using a surface plasmon resonance based activity assay. We hope that this study will stimulate utilization of such higher generation dendrons for enhancing thermal stability of mAbs. Keywords: Monoclonal antibody, stability, excipients, dendrons, size exclusion chromatography, haemolytic activity

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Graphical Abstract



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Introduction

Monoclonal antibodies (mAbs) are one of the most important and widely studied class of therapeutic proteins. These products are widely used in the treatment of a variety of diseases, including cancers and inflammatory diseases.1-2 In 2014, there were 47 mAbs approved commercially by the various regulatory agencies in US and Europe at the approval rate of four new products per year.3 Despite their wider use, mAbs are very sensitive to the minute physicochemical changes in the environment. A number of physical and chemical factors, during manufacturing or storage processes, can cause their aggregation, fragmentation, oxidation, deamidation, reduction, and truncation.4-5 These degradations not only reduce the therapeutic efficiency of mAbs but may also cause an immunogenic response during therapy.6-7 So while developing an effective

drug product, all physicochemical parameters such as pH,

temperature, buffer composition, protein concentration, ionic strength, and shear rate need to be optimized and maintained.8-10

Over the decades, excipients such as sucrose, sorbitol, mannitol and trehalose have been utilized to improve shelf life of mAb based formulations.11-14 However, concerns related to stability of these products remain, especially with respect to stability at and above room temperature. At present, all biotech therapeutics require refrigeration during storage to avoid degradation and preserve biological activity. The optimal storage temperature ranges from -70oC for long term usage to 4oC for short term usage.15-16 This can be a challenge, particularly in developing and underdeveloped economies of the world, where maintaining these storage conditions may not be feasible. There is an urgent need to develop novel formulations that can offer product stability at room and higher temperatures.

Dendrimers are a special class of synthetic molecules having branched ‘tree4 ACS Paragon Plus Environment

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like’ architectures. Dendrons are smaller units of dendrimers. Due to their discrete

molecular

weight,

tunable

size,

shape,

and

multivalency,

dendrons/dendrimers are identified as suitable candidates for drug delivery, transfectants and as antimicrobial agents.17 However, their use as excipients in mAb formulations has not been widely explored.18-20

In the studies presented in this paper, we have chosen lysine (Lys) amino acid as the building block for the synthesis of dendrons in view of their known interactions with antibodies.21-22 We have earlier reported on their impact on protein refolding.23 Since mAb aggregation at high temperatures is primarily attributed to conformational destabilization or partial to complete unfolding24-25, this served as the motivation for the present investigation.

Results and Discussion

The protected dendrons L1, L3, L5 and deprotected dendrons L2, L4, L6 were prepared in a divergent strategy following the experimental regimen that has been reported previously (Scheme 1).26-28 The purity of dendrons was confirmed by high resolution mass spectrometry (HRMS) (Figure S2, ESI†) and high performance liquid chromatography (HPLC) (Figure S3, ESI†).

Optimization of Dendron:mAb Molar Concentration

In order to study the effect of dendrons on thermal stability of mAbs, first the amount of dendrons that is to be added to give maximum recovery of monomer content was optimized by monitoring the percentage of monomer in formulation as a function of time at three different ratios of molar concentrations (mAb: dendrons) viz. 1:1, 1:2, 1:3 and it was found that all three dendrons exhibited optimal reduction in aggregation and fragmentation when added in an equivalent amount to mAb (molar ratio). This is evident from SEC 5 ACS Paragon Plus Environment


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Scheme 1: Divergent Synthesis of Peptide Dendrons. data presented in Table 1 and Figure 1 and S4, ESI†. In view of these results, all subsequent studies were done with 1:1 molar ratio (mAb:dendrons). Fragmentation refers to hydrolysis or cleavage at the hinge region, resulting in creation of fragment species of 97 KDa and 47 KDa molecular weights. Dendrons L2

L4

L6

Concentration of mAb:dendron 1:1 1:2 1:3 1:1 1:2 1:3 1:1 1:2 1:3

% Monomer

% Aggregate

% Fragment

78.7 61.9 57.1 80.2 74.7 70.5 83.3 79.4 78.9

13.2 2.2 0.0 13.7 2.2 1.9 9.2 11.6 13.4

8.1 35.9 42.9 6.1 23.1 27.7 7.4 13.6 18.1

Table 1: Effect of dendrons of different generations on mAb stability at 55 oC after 11 days at varying concentrations. Aggregation and fragmentation have been measured by SEC. 6 ACS Paragon Plus Environment

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At elevated temperatures, mAbs undergo degradation primarily through aggregation and fragmentation.25,29 Thus, monitoring of aggregates and fragments in the presence of dendrons was performed to compare the effectiveness of the various dendrons in stabilizing the mAb at 55oC. The results are tabulated in Table 1 (Figure 1, S4, S5 and S6, ESI†). A careful analysis of these results reveals that in case of L2 and L4, as the molar concentration of dendrons increases, the percentage of aggregation decreases and is 0.00% in case of L2 (at ratio 1:3). This was better than the observations with the third generation dendron L6. However, it was observed that at higher temperatures both aggregation and fragmentation can occur and hence both need to be monitored during evaluation of product stability.30 While L2 (1:3) results in least aggregation, fragmentation is quite significant under these conditions.

Figure 1: Percentage of monomer, aggregates and fragments in mAb formulation (5mg/ml) at 55⁰C with a) Control and PS 80 b) L6 (1:1), b) L6 (1:2) and c) L6 (1:3). 7 ACS Paragon Plus Environment


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On the other hand, in the case of L6, unlike L2 and L4, aggregation increases as the molar concentration of L6 increases and is least in a 1:1 formulation. This is better than the corresponding L2 and L4 (1:1) formulations. Moreover, in case of L6 (1:1), percentage of recovered monomer content is highest than in any other formulation. Hence, it can be inferred that third generation dendron L6 is the optimal thermal stability enhancer for mAb formulation when used in 1:1 mAb:dendron molar concentration ratio.

Comparison of L6 Dendron with PS 80 In the next study, the performance of L6 as a mAb stabilizer was compared to that of the commonly used excipient, Polysorbate 80 (PS 80). It was found that percent monomer loss rate was 3.7 times higher with PS 80 than with

Figure 2: Overlay of SEC chromatograms for mAb formulation (5mg/ml) at 55⁰C with a) PS 80 b) L2 (1:1), b) L4 (1:1) and c) L6 (1:1) showing comparative decrease in monomer content.

dendrons (Figure 2and S7, ESI†). Further, it was observed that loss of monomer 8 ACS Paragon Plus Environment

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in PS 80 formulation due to precipitation was 44.28% while in L6 (1:1) it was just 1.52% (Figure 2). Thus, the results indicate the superiority of L6 dendron over PS 80 as a mAb stabilizer. It can be concluded that L6 interacts with mAb and hence prevents it from forming soluble as well as insoluble aggregates. In other words, L6 provides colloidal stability to mAb at elevated temperatures. Also, we should highlight that excipients like PS 80 and L6 dendrons are used well below their CMC and hence they do not aggregate at high temperatures (55⁰C). We verified this by performing separate negative control experiments with samples containing only the respective excipients (PS 80 and L6 dendron) without any mAb. No significant signal or peak at 280 nm was observed (Figs. S16 and S17, ESI†).

Secondary Structure Analysis In addition to enhancing colloidal stability, an ideal excipient should also preserve the conformational stability of protein.31 The impact of dendrons on conformational stability of mAbs was examined by CD spectroscopy (190-250 nm). mAbs contain large number of β-sheets. If aggregate content increases, βsheet content increases as well and this can be characterised by a minimum at 218nm in the CD spectrum.32 The CD spectra of formulations with PS 80 and dendrons L2, L4, L6 show formation of aggregates (Figure 3a). In order to study the comparative effect of dendrons, the mean residue ellipticity (MRE) values at 218 nm obtained from CD spectra were further plotted against the incubation time (Figure 3b). It is seen that L6 yields least fluctuations in MRE values as compared to PS 80, L2, and L4. Thus, it can be inferred L6 is the most effective in preserving the secondary structure of mAb and thereby enhancing its conformational stability. The other important observation that can be drawn from CD data is that the aggregation formed in presence of dendrons is somewhat more stable than that in presence of PS 80 (Figure 3a and 3b). 9 ACS Paragon Plus Environment


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Figure 3: CD spectroscopy of samples. a) CD spectra of mAb formulations at 5mg/ml with PS 80, L2, L4 and L6 (1:1). b) MRE values at 218 nm versus incubation time for formulation at 5 mg/ml with PS 80 and formulation with 1:1 dendrons at 55⁰C.This also correlates with the SEC data and maybe attributed due to the chemical interactions of dendrons with aggregated mAb molecules which provide stability to them and thus prevent further fragmentation.33 Moreover, the CD data is in agreement with SEC data where aggregate degradation into fragments was observed with time in formulation containing PS 80 (Figures 2 and S7, ESI†).

SDS PAGE Analysis The results obtained from SEC were further supported by the observations from non-reducing SDS PAGE. As seen in Figure 4, four major bands are observed in all the samples loaded in the wells (2-3). The band at the top corresponds to the aggregate with size larger than 200 KDa. The second band is that for the monomer with a molecular weight of around 150KDa. The next band corresponds to around 100 KDa and the last band is slightly below 50 KDa.


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Figure 4: SDS PAGE analysis of stressed mAb samples at 5 mg/ml. Lane 1 indicates the molecular weights of standard in KDa; Lane 2: mAb sample with PS 80 stressed at 55 ᵒC for 6 days; Lane 3: mAb sample with L6 dendron stressed at 55 ᵒC for 6 days; Lane 4: mAb sample without any excipient stressed at 55ᵒC for 13 days. The mAb sample was stressed for 13 days at 55ᵒC without any excipient and was mainly composed of fragments with size equivalent to 47 KDa and 34 KDa.

Size based DLS Analysis For gaining further insight into mAb aggregation, DLS measurements were also performed.34 The percentage intensity vs. size plot indicates that the broad maxima in range of 50-80 nm decreases from L2 to L6 (Figure 5a and S8, ESI†). These maxima correspond to aggregates formed while the sharp maxima in the range of 10-15 nm correspond to the monomer content. As we move from L2 to L6 (1:1), the aggregate content decreases and monomer content increases. These observations are in agreement with those made from SEC (Table 1). The DLS data further confirms the superior performance of L6 as mAb stability enhancer when compared to other dendrimers examined as well as the PS 80 (Figure 5a, S9 and S10, ESI†). Since the data provide by DLS is mono-modal in nature, to confirm whether the sample contains various aggregate species, 11 ACS Paragon Plus Environment


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nano particle tracking analysis (NTA) of the samples was performed (Figures 5c and 5d). NTA analysis of control sample without any excipient and heated at 55⁰C shows formation of larger aggregates ranging from 160 nm-340 nm with concentrations as high as 3 x 107 number/ml. On the other hand, NTA analysis of the sample incubated with L6 dendron showed presence of particles of similar size range but at significantly lower concentrations (1x 107 number/ml). DLS, due to its advantage of high throughput and operability and small sample requirement, is widely used for determination of intermolecular interactions via calculation of the interaction parameter, kd, based on the concentration dependence of diffusion coefficient using the following equation35: D(c) = Do (1+kdC) Where, D(c) is the measured diffusion coefficient, Do is diffusion coefficient at infinite dilution and c is the concentration of mAb (µM). Value of kd denotes more repulsive forces between protein molecules and hence lesser disposition towards aggregate formation which further implies greater stability of protein in formulation. Further, kd is the measure of extent of intermolecular interactions and hence it indirectly measures the extent of aggregation. It is observed in Figure 5b (Figure S11 and S12, ESI†) that kd is highest (least negative) in case of L6 followed by L4 and L2, respectively. Thus mAb has higher intermolecular repulsive interactions in presence of L6 dendron as compared to L2 and L4 and therefore are less prone to aggregation. Hence kd profile also supports our key observation that L6 augments mAb thermal stability most significantly among all the three generation of dendrons examined in this study.


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Figure 5: Size based analysis of mAb samples at 5 mg/ml for confirmation of colloidal stability. a) DLS intensity vs. size plot for mAb formulation with PS 80 and 1:1 dendrons on 9th day; b) Interaction parameter profile for dendrons indicative of mAb intermolecular interactions; c) DLS intensity vs. size plot for control mAb; d) NTA analysis of control sample and mAb with L6 dendron (1:1) at 7th day.

In-vitro Binding Assay mAbs along with these dendrons were able to retain their activity and it was confirmed by performing SPR based assay for standard formulations with PS 80 as well as formulations with K7 dendrons in the best working ratio of 1:1 (Figure 6). This can be clearly observed in Table 2, where % change in value of rate constant (KD) is negligible amongst samples with L6 (1:1) Dendron and samples with PS 80 respectively. Also the % change in Rmax values obtained from the SPR analysis for both samples (mAb with PS80 and mAb with L6 (1:1)) is within acceptable range which confirms that there is no loss in activity of monoclonal antibody on addition of L6 dendron. Although the sensorgrams in the experiments were able to be fitted by both univalent and bivalent models, 13 ACS Paragon Plus Environment


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they were better fitted by the 1:1 than bivalent analyte model. The values of kinetic parameters obtained from in vitro binding assay has been shown in Table 3. It is evident that values of Ka, Kd and KD do not change significantly on addition of L6 dendron to the formulation and it can be concluded that the presence of dendrons does not impact the activity of the mAb. Percentage Monomer Aggregate Fragment Haemolysis Change in Rmax Change in KD

Control without Excipient 48.8 0.9 50.4 -

PS80

Dendron L6(1:1)

62.6 0.0 37.4 5.2 2.24 0.0

83.3 9.2 7.4 2.8 -5.42 0.0

Table 2: Comparison of commercially used excipient PS 80 with third generation dendron L6.

Samples mAb mAb with 0.02% PS 80 mAb with L6 dendron (1:1)

Ka (1/Ms) 1.26 x 106 1.17 x 106 1.07 x 106

Kd(1/s) 1.74 x 10-3 1.65 x 10-3 2.2 x 10-3

KD(M) 1.35 x 10-9 1.41 x 10-9 2.11 x 10-9

Table 3: Comparison of Kinetic parameters (Ka, Kd, KD) of different samples.

Figure 6: Kinetic analysis of a) Control without excipient, b) mAb with PS 80 and c) mAb with L6 Dendron (1:1) using SPR. Biocompatibility Assay Biocompatibility of these dendrons was confirmed by performing the haemolytic activity test36 for the formulations with PS 80 as well as with 14 ACS Paragon Plus Environment

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dendrons in different ratios on initial and last day (Figure 7a and 7b). Bar graph in Figure 7b clearly depicts that percentage haemolysis in formulation with dendrons is in the range of 2.6-2.8% and is negligible. PS 80 exhibited a haemolysis 1.8 times higher than L6 (Table 2) indicating that L6 containing formulations are safe for use in commercial formulations. Effect of dendrons on red blood cells (RBC’s) was visualised by using confocal microscopy (Figure S13 and S14, ESI†). It can be seen that in case of PS 80 there is bit shrinkage of RBC’s compared to the control sample while in case of L6 no such effect is observed.

Figure 7: a) Confocal microscopic images of RBC’s in blank, formulation with PS 80 and dendron L6 b) Bar graph showing the percentage haemolysis by PS 80 and dendrons on initial day and 11th day. 15 ACS Paragon Plus Environment


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Demonstration of Effectiveness of L6 Dendron as a Stabilizer for a Second mAb (Anti VEGF) To demonstrate the broader applicability of L6 dendrons as stabilizers for mAb formulations, experiments were performed using a second mAb (Anti VEGF) and the results are presented in Figures 8a and 8b and Table 4. It is evident that anti VEGF mAb at 10 mg/ml of concentration is more stable in presence of L6 dendrons and exhibits reduced aggregate formation (1.31%) as compared to the control sample (4.32%). Also, L6 dendrons help in stabilizing anti VEGF mAb against fragmentation. There is an insignificant increase in fragmentation of anti VEGF mAb in presence of L6 dendrons (0.18%) as compared to the control sample (1.27%). The increased stability of anti VEGF mAb in presence of L6 dendron is also confirmed by examining the samples for aggregates of various sizes by DLS and NTA.

Figure 8: Stability of anti VEGF mAb at 10 mg/ml in presence of L6 dendron. SEC analysis for aggregation and fragmentation in a) Control mAb and b) mAb 16 ACS Paragon Plus Environment

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with L6 dendron. Size based analysis of aggregation using c) DLS for control mAb at 0 day and d) NTA for control mAb at 7th day and mAb with L6 dendron at 0th and 7th day.

Sample mAb VEGF)

% Monomer (Anti 92.86%

% Aggregation

% Fragmentation

3.51%

3.62%

7.83%

4.89%

5.31%

0.53%

6.62%

0.71%

control

0day mAb

(Anti 90.17%

VEGF) Control 7 day mAb

(Anti 94.16%

VEGF) with L6 dendron 0day mAb

(Anti 92.67%

VEGF) with L6 dendron 7 day

Table 4: Effect of L6 dendron on anti VEGF mAb stability at 55oC after 7 days. Aggregation and fragmentation have been measured by SEC. Conclusions Third generation peptide dendrons provided colloidal as well as conformational stability to mAb formulation at 55oC as demonstrated by a host of analytical techniques such as Size Exclusion Chromatography (SEC), Dynamic Light Scattering (DLS), and Circular Dichroism (CD). The bio-compatibility of these dendrons was confirmed by haemolytic activity tests. Non-interference of the dendrons with the activity of the mAb was confirmed using a surface plasmon resonance based activity assay. We hope that this study will stimulate utilization 17 ACS Paragon Plus Environment


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of such higher generation dendrons for enhancing thermal stability of mAbs. The C-terminal alkyne group in the dendrons can be dimerized for the synthesis of corresponding dendrimers with increased number of hydrophilic amine groups. Such dendrimers are likely to be potential excipients for mAb formulations and will be focus of our future studies.

Materials and Methods Feed Material An IgG1 antibody designated as anti-CD6 monoclonal antibody of immunoglobulin (Ig) G1 isotype that targets the SRCR1 (Scavenger Receptor Cysteine Rich) domain of CD6 was obtained from a major domestic biopharmaceutical producer. This mAb had a pI of 8.5. The mAb was stored at a concentration of 28 mg/ml in 15 mM phosphate and 150 mM sodium chloride buffer at 4 ºC. Another IgG1 antibody, designated as anti-VEGF monoclonal antibody of immunoglobulin (Ig) G1 isotype, that targets the soluble VEGF receptor and blocks angiogenesis was obtained from another major domestic biopharmaceutical producer. This mAb had a pI of 8.2 and was stored at a concentration of 15 mg/ml in 20 mM phosphate and 200 mM sodium chloride buffer at 4 ºC.

Chemical Synthesis of Dendrons All amino acids used were of L-configuration. Unless otherwise stated, all reagents were used without further purification. All solvents employed in the reactions were distilled or dried from appropriate drying agent prior to use. Amino acid L-Lysine was purchased from SRL India. Reactions were monitored wherever possible by thin layer chromatography (TLC). Purification of compounds was done by silica gel column chromatography. Silica gel G (Merck) was used for TLC and column chromatography was done on silica gel (100-200 mesh) columns, which were generally made from slurry in hexane, 18 ACS Paragon Plus Environment

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hexane/ethyl acetate or chloroform. Analytical HPLC was carried out using Eclipse XDB-C18 column and acetonitrile/water as the solvent system (Figure S2, ESI†, HPLC profiles of various dendrons). Detailed procedures have been described earlier.26-28 General procedure for the peptide coupling reaction To an ice-cooled and well stirred solution of N-protected amino acid (1.0 mmoles) in dry dichloromethane, was added N-hydroxysuccinimide (1.2 mmol), dicyclohexylcarbodiimide (DCC) (1.2 mmol) and stirred for 10 min. To this mixture was added an amine component (1.2 mmol) in dichloromethane and triethylamine (1.2 mmol). The reaction mixture was stirred overnight, filtered and washed the filtrate with 0.2 N H2SO4, water and finally with saturated aqueous NaHCO3 solution. The organic layer was separated, dried over anhydrous Na2SO4, filtered and evaporated. Silica gel column chromatographic purification yielded the products in ~ 70-92% yields.

General procedure for the Boc-Deprotection To an ice-cooled solution of the Boc-protected compound (1mmol) was added 25 % solution of trifluroacetic acid TFA (40 mmol) in dry dichloromethane and stirred at room temperature for 3h. The reaction mixture was subjected to high vacuum, redissolved in ethylacetate/dichloromethane and washed with sodium carbonate. The organic layer was dried over anhydrous Na2SO4 and evaporated. All the prepared dendrons were characterized by 1H NMR, 13C NMR, IR and HRMS.26-28 Purity of the compounds was analyzed by analytical HPLC (Figure S2, ESI†) (Agilent Technologies 1200 series, Santa Clara, USA) equipped with Eclipse XDB-C18 reversed phase column using acetonitrile/water as the solvent system.

Reagents 19 ACS Paragon Plus Environment


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mAb A was formulated in 15 mM phosphate, 150 mM sodium chloride and 0.02% PS 80, pH 6.5. For stability studies with excipients like PS 80 and dendrons, stock solutions were prepared. Final protein formulation was obtained by adding adequate amount of protein, pure buffer and excipient solution, so that the final protein concentration in the sample solution is 33.33 µM. The dendrons were added to these formulation samples in the molar concentration ratio of 1:1, 1:2 and 1:3. All buffers were filtered using a 0.22 µm nylon membrane filter (PALL Life Sciences, Port Washington, NY, USA) and then degassed. All chemicals used for formulation preparation were of Analytical grade procured from Sigma Aldrich (Bengaluru, Karnataka, India). For characterization in SEC, mobile phase composed of chemicals of analytical grade. Sample preparation First generation L1, second generation L3, and third generation L5 Lys-based dendrons, each containing tert-butyloxy carbonyl (Boc) as protecting group were synthesized (Figure 7). While the mAb formulation is in an aqueous medium, the presence of these hydrophobic Boc groups was a deterrent in solubilization of the dendron in water. Hence, the dendrons were modified by removing the Boc groups and generating hydrophilic deprotected dendrons L2, L4, L6 (Figure 9). These modified dendrons were added to mAb solutions and the formulation was subjected to accelerated thermal stress for 11 days at 55oC. The stability of these dendrons with mAb formulations have been tested at both room temperature (30 oC) (Table S1 and Figure S1, ESI†) and higher temperature (55oC). Since at room temperature of 30oC, these formulations were quite stable, so accelerated degradation studies at higher temperature of 55oC were performed to check the stability of these mAb formulations with dendrons. The stability provided by dendrons to mAb at this temperature was monitored by several high-resolution, orthogonal analytical techniques including size


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exclusion chromatography (SEC), dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy.30,32,36-37 Further, the haemolytic activity test was performed to confirm the biocompatibility of these dendrons.38 The biological activity of these dendrons with mAb formulations was tested using surface plasmon resonance (SPR) based method.

Figure 9: Chemical structures of dendrons L2 (First generation); L4 (Second generation); L6 (Third generation). Blue color highlights hydrophilic amine group. To obtain protein in desired formulation buffer and at 5 mg/ml concentration, buffer exchange using a Sephadex G-25 resin (GE Healthcare Biosciences, Pittsburgh, PA, USA) packed into a Tricon™ column (100 × 10 mm) was performed. Concentrated protein was collected after buffer exchange from Äkta Purifier (GE Healthcare Biosciences, Pittsburgh, PA, USA) by observing absorbance at 280 nm. Protein concentration was measured to be 5 mg/ml at 280 nm using a SpectraMax M2e Multimode Microplate Reader (Molecular 21 ACS Paragon Plus Environment


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Devices, Sunnyvale, CA, USA). Extinction coefficient of mAb was 1.45 ml* mg-1cm-1. Stress method: Storage at elevated temperature To test the effect of high temperature on mAb stability, solutions at 5mg/ml were stored in 2ml centrifuge tube at 55ºC for 11 days. At specific time points, fixed volume of samples were taken out for analysis by various techniques. All experiments were performed in duplicate and average values have been reported. Analytical Methods: Size-Exclusion chromatography (SEC) For characterization, SuperdexTM 200 (300 x 10 mm, pore size 8.6µm) high resolution column operated at 25 ºC was used. The column was connected with Agilent 1200 HPLC series (Agilent Technologies, Santa Clara, USA) consisting of a quaternary pump with a degasser, an auto sampler with a cooling unit, and a variable wavelength detector (VWD). Before analysis, 100 µl samples was taken out and centrifuged at 10000 g for 1 min to remove insoluble aggregates. Mobile phase of composition 50 mM phosphate buffer, 300 mM sodium chloride, and 0.05% sodium azide at pH 7.0 was filtered with a 0.22- µm nylon membrane filter and degassed prior to use. Isocratic elution was performed for 45 min at a flow rate of 0.5 ml/min. Measurements were done in triplicates and averaged peak area of aggregate, monomer and fragment content was obtained. Circular Dichroism (CD) measurements The circular dichroism spectra of mAb in the presence and absence of dendrons were obtained to determine the changes in secondary structure. Far-UV CD spectra analysis was performed in Jasco J- 815 CD spectrometer (Jasco Analytical Instruments, Mary's Court, USA). Sample concentration was kept at 0.2 mg/ml and wavelength in the range of 190–250 nm was used to obtain spectra. For spectral measurements, 300 µl of sample solution was taken in 22 ACS Paragon Plus Environment

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quartz cuvette (1 mm path length) at 20°C and an average of five scans was taken. CD spectra of the buffer solutions were subtracted from the sample spectra before conversion to absolute CD values. The mean residual ellipticity values (MRE) at wavelength λ ([θ]MRW, λ) were calculated using the mean residual weight (MRW) for the antibody as follows32: [θ]MRW,λ =

(1)

Where θλ is the observed ellipticity (degrees) at wavelength λ, d is the path length (cm), and c is the concentration (g/ml).

Dynamic Light Scattering (DLS) measurements A Zetasizer Nano ZS 90 (Malvern Instruments, UK) particle size analyzer with temperature control fitted with a 633-nm He-Ne laser was used to determine the hydrodynamic radii (RH) of the samples by using dynamic light scattering. The instrument used had a wide size range measuring capability from 0.3 nm to 5000 nm. 1.2 ml of mAb solution was taken in a 3 ml plastic disposable cuvette which was properly washed with ethanol to ensure no contamination from dust particles. The protein sample was filtered with 0.4 µm membrane filter (Pall Corp., USA). Sample concentration taken for the DLS measurement was 1 mg/ml which was measured by recording UV absorbance at 280 nm. From the RH value obtained, diffusion coefficient, D had been determined using Stokes-Einstein39 equation:

(2)

Where KB is defined as Boltzmann constant, T is absolute temperature (25°C) and ηs represents solvent viscosity (0.8mPa.s). All the scattered intensities were recorded at a fixed angle of 90°. 23 ACS Paragon Plus Environment


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Nano Particle Tracking Analysis (NTA) NTA analysis was performed with a NanoSight NS 300 (Malvern Instruments UK) fitted with a sample chamber with a 532 nm laser and sCMOS camera. The samples were introduced in the sample chamber with sterile syringes until the nozzle was completely filled with the liquid. The software used for data capturing and analysis was NTA 3.1 Build 3.1.54. The video capture time used was 90 s due to the sample’s high polydispersity. Six measurements of each sample were taken and mean was obtained. Stressed formulations were diluted 250 folds with buffer before each measurement. SDS-Polyacrylamide Gel Electrophoresis (PAGE) The molecular weights of various aggregates, monomer, and fragments species that were formed was also confirmed by performing SDS- PAGE under nonreducing conditions. About 4µg of mAb sample was loaded in each well. The samples were incubated at 90°C for 5 minutes prior to loading. Electrophoresis was carried out at a constant current of 20 mA with 1X Tris-Glycine SDS running buffer. After electrophoresis, gel was stained using standard silver staining protocol. These silver stained gels were scanned and analysed using GS-800 densitometer equipped with Quantity One software (Bio-Rad Laboratories, Hercules, CA). The approximate molecular weights of different samples present in the various bands were estimated by visually comparing them to the bands that were present in the protein marker.

Interaction parameter (kd) determination Diffusion coefficient values of each sample were obtained by DLS measurement using Zetasizer Nano ZS 90 software at same mAb concentration of 33.33 µM (5 mg/ml) but varying dendrons concentration of 33.33 µM, 66.66 µM and 133.32 µM. Diffusion coefficient readings were obtained in triplicates which were averaged. Interaction parameter kd was obtained from the slope of 24 ACS Paragon Plus Environment

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the Diffusion coefficient D(c) as a function of dendrons concentration whose straight line equation is35:

= +

(3)

Where, = Measured diffusion coefficient of sample, = Diffusion coefficient at infinite dilution (buffer), = concentration Haemolytic activity test Haemolytic activity was checked for mAb formulations by a reported in-vitro method.38 Freshly collected blood sample was washed three times with 10 mM phosphate saline (0.9% NaCl) maintaining isotonic condition by centrifuging at 1500 rpm for 10 min at 4ºC. Pure RBC pellets obtained after washing was resuspended in 10 mM phosphate saline (0.9% NaCl), pH 7.4. 100 µl of formulation at 5 mg/ml was added to 100 µl of RBC resuspension and incubated at 37ºC and 100 rpm. After 30 min, suspension was centrifuged at 1500 rpm and supernatant

collected

was

analyzed

for

haemoglobin

release

by

spectrophotometric determinations at λ max 416 nm using SpectraMax M2 (Molecular Devices, LLC, California) in 96 well-plate. To obtain 0 % and 100 % hemolysis, RBC suspension in 10 mM phosphate saline (0.9% NaCl) and Triton-X (positive control) was performed, respectively. Following equation was used to determine % haemolysis: ()*+,-./0 1(234567

% !"#$%&'& = (

)*+,-./0 1(8997*

× 100%

(4)

Confocal microscopic Images of RBC’s were taken in Olympus Fluoview FV1000 at 60X magnification. Surface Plasmon Resonance based activity assay The purified recombinant CD6 protein (purchased from R&D Systems) was diluted with 10 mM sodium acetate (pH 5.0) and injected at 25 C ̊ and was 25 ACS Paragon Plus Environment


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immobilized onto a CM5 biosensor chip (Biacore, GE Healthcare) using an amine coupling kit (Biacore) at relatively low densities (1000 resonance units) to avoid mass transport limitation. The reference cell was treated with Nhydrox- ysuccinimide (NHS) /1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and ethanol amine using an amine coupling kit. The running buffer, HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% Surfactant P20 [pH 7.4]) (Biacore), was allowed to flow at 30 ml/min. The injections were performed using contact time 60s and dissociation time, 180s. Six concentrations (0.5 to 16nM) of mAb were then injected. Because injection at higher concentrations caused nonspecific binding to flow cells, the analysis for the affinity of therapeutic proteins using sensorgrams obtained at the concentrations at which nonspecific binding was not observed. For regeneration, the regeneration buffer (10Mm Glycine –HCl (pH 2.5) was injected for 4 min. Kinetic constants were calculated from the sensorgrams using the 1:1 fit model of Biacore X100 Evaluation Software 2.0.1. Although the sensorgrams in the experiments were able to be fitted by both models, they were better fitted by the 1: 1 than bivalent analyte model.

Associated Content: Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

.

SI contains data pertaining to HRMS and HPLC profiles of dendrons, SEC data at 30 ⁰C, SEC data at 55 ⁰C , DLS data, Hemolytic activity data with confocal microscopic images. 26 ACS Paragon Plus Environment

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Notes The authors declare no competing financial interest. Abbreviations: mAb - Monoclonal Antibody SEC-HPLC – Size Exclusion Chromatography High Performance Liquid Chromatography HRMS- High Resolution Mass Spectrometry DLS - Dynamic Light Scattering CD- Circular Dichroism MRE – Mean Residual Ellipticity

Acknowledgements This work was supported by Department of Science and Technology, New Delhi (DST). We thank DST-FIST for mass spectral facility at IITD. SD thanks DST for the INSPIRE fellowship. GPM thanks University Grants Commission (UGC), New Delhi for the fellowship. We also acknowledge funding from DBT Centre

of

Excellence

for

Biopharmaceutical

Technology

(number

BT/COE/34/SP15097/2015). We also thank Malvern Instruments, UK for providing us the access to the NTA facility for particle size analysis. References 1. Beck, A., Wurch, T., Bailly, C., and Corvaia, N. (2010) Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev.

Immunol. 10, 345–352. 2. Farhadfar, N., and Litzow, M. R. (2016) New monoclonal antibodies for the treatment of acute lymphoblastic leukemia. Leuk. Res. 49, 13–21. 27 ACS Paragon Plus Environment


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3. Ecker, D. M., Jones, S. D., and Levine, H. L. (2015) The therapeutic monoclonal antibody market. mAbs 7, 9–14. 4. Wang, W. (1999) Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int. J. Pharm. 185, 129–188. 5. Frokjaer, S., and Otzen, D. E. (2005) Protein drug stability: a formulation challenge. Nat. Rev. Drug Discov. 4, 298–306. 6. Daugherty, A. L., and Mrsny, R. J. (2006) Formulation and delivery issues for monoclonal antibody therapeutics. Adv. Drug Deliv. Rev. 58, 686–706. 7. Joubert, M. K., Hokom, M., Eakin, C., Zhou, L., Deshpande, M., Baker, M. P., Goletz, T. J., Kerwin, B. A., Chirmule, N., Narhi, L. O., and Jawa, V. (2012) Highly Aggregated Antibody Therapeutics Can Enhance the in Vitro Innate and Late-stage T-cell Immune Responses. J. Biol. Chem.

287, 25266–25279. 8. Joshi, V., Shivach, T., Kumar, V., Yadav, N., and Rathore, A. (2014) Avoiding antibody aggregation during processing: Establishing hold times. Biotechnol. J. 9, 1195–1205. 9. Schreiber, G. (2002) Kinetic studies of protein–protein interactions. Curr.

Opin. Struct. Biol. 12, 41–47. 10. Singla, A., Bansal, R., Joshi, V., and Rathore, A. S. (2016) Aggregation Kinetics for IgG1-Based Monoclonal Antibody Therapeutics. AAPS J. 18, 689–702. 11. Manikwar, P., Majumdar, R., Hickey, J. M., Thakkar, S. V., Samra, H. S., Sathish, H. A., Bishop, S. M., Middaugh, C. R., Weis, D. D., and Volkin, D. B. (2013) Correlating Excipient Effects on Conformational and Storage Stability of an IgG1 Monoclonal Antibody with Local Dynamics as Measured by Hydrogen/Deuterium-Exchange Mass Spectrometry. J.

Pharm. Sci. 102, 2136–2151.


Page 29 of 33

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


12. Serno, T., Carpenter, J. F., Randolph, T. W., and Winter, G. (2010) Inhibition of Agitation‐Induced Aggregation of an IgG‐Antibody by Hydroxypropyl‐β‐Cyclodextrin. J. Pharm. Sci. 99, 1193–1206. 13. Cheng, W., Joshi, S. B., He, F., Brems, D. N., He, B., Kerwin, B. A., Volkin, D. B., and Middaugh, C. R. (2012) Comparison of HighThroughput Biophysical Methods to Identify Stabilizing Excipients for a Model IgG2 Monoclonal Antibody: Conformational Stability and Kinetic Aggregation Measurements. J. Pharm. Sci. 101, 1701–1720. 14. Thakkar, S. V., Joshi, S. B., Jones, M. E., Sathish, H. A., Bishop, S. M., Volkin, D. B., and Russell Middaugh, C. (2012) Excipients Differentially Influence the Conformational Stability and Pretransition Dynamics of Two IgG1 Monoclonal Antibodies. J. Pharm. Sci. 101, 3062–3077. 15. Mazid, R. R., Vijayaraghavan, R., MacFarlane, D. R., Cortez-Jugo, C., and Cheng, W. (2015) Inhibited fragmentation of mAbs in buffered ionic liquids. Chem Commun 51, 8089–8092. 16. Paul, M., Vieillard, V., Jaccoulet, E., and Astier, A. (2012) Long-term stability of diluted solutions of the monoclonal antibody rituximab. Int. J.

Pharm. 436, 282–290. 17. Wu, L., Ficker, M., Christensen, J. B., Trohopoulos, P. N., and Moghimi, S. M. (2015) Dendrimers in Medicine: Therapeutic Concepts and Pharmaceutical Challenges. Bioconjug. Chem. 26, 1198–1211. 18. Sorokina, S. A., Stroylova, Y. Y., Shifrina, Z. B., and Muronetz, V. I. (2016) Disruption of Amyloid Prion Protein Aggregates by Cationic Pyridylphenylene Dendrimers: Disruption of Amyloid Prion Protein Aggregates. Macromol. Biosci. 16, 266–275. 19. Nowacka, O., Shcharbin, D., Klajnert-Maculewicz, B., and Bryszewska, M. (2014) Stabilizing effect of small concentrations of PAMAM dendrimers at the insulin aggregation. Colloids Surf. B Biointerfaces 116, 757–760. 29 ACS Paragon Plus Environment


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Page 30 of 33

20. Shukla, D., Schneider, C. P., and Trout, B. L. (2011) Effects of PAMAM Dendrimer Salt Solutions on Protein Stability. J. Phys. Chem. Lett. 2, 1782–1788. 21. Haridas, V., Sharma, Y. K., Sahu, S., Verma, R. P., Sadanandan, S., and Kacheshwar, B. G. (2011) Designer peptide dendrimers using click reaction. Tetrahedron 67, 1873–1884. 22. Hüttl, C., Hettrich, C., Riedel, M., Henklein, P., Rawel, H., and Bier, F. F. (2015) Development of Peptidyl Lysine Dendrons: 1,3-Dipolar Cycloaddition for Peptide Coupling and Antibody Recognition. Chem.

Biol. Drug Des. 85, 565–573. 23. Dubey, P., Gautam, S., Kumar, P. P. P., Sadanandan, S., Haridas, V., and Gupta, M. N. (2013) Dendrons and dendrimers as pseudochaperonins for refolding of proteins. RSC Adv. 3, 8016. 24. Mahler, H.-C., Friess, W., Grauschopf, U., and Kiese, S. (2009) Protein aggregation: Pathways, induction factors and analysis. J. Pharm. Sci. 98, 2909–2934. 25. Hawe, A., Kasper, J. C., Friess, W., and Jiskoot, W. (2009) Structural properties of monoclonal antibody aggregates induced by freeze–thawing and thermal stress. Eur. J. Pharm. Sci. 38, 79–87. 26. Haridas, V., Lal, K., and Sharma, Y. K. (2007) Design and synthesis of triazole-based peptide dendrimers. Tetrahedron Lett. 48, 4719–4722. 27. Haridas, V., Sharma, Y. K., and Naik, S. (2009) Multi-Tier Dendrimers with an Aromatic Core. Eur. J. Org. Chem. 2009, 1570–1577. 28. Haridas, V., Sharma, Y. K., Creasey, R., Sahu, S., Gibson, C. T., and Voelcker, N. H. (2011) Gelation and topochemical polymerization of peptide dendrimers. New J Chem 35, 303–309. 29. Vlasak, J., and Ionescu, R. (2011) Fragmentation of monoclonal antibodies. mAbs 3, 253–263.


Page 31 of 33

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


30. Berkowitz, S. A., Krull, I., and Rathore, A. (2015) Challenges in the Determination of Protein Aggregates, Part II. LCGC North America 33, 478-489. 31. Goldberg, D. S., Bishop, S. M., Shah, A. U., and Sathish, H. A. (2011) Formulation Development of Therapeutic Monoclonal Antibodies Using High-Throughput Fluorescence and Static Light Scattering Techniques: Role of Conformational and Colloidal Stability. J. Pharm. Sci. 100, 1306–1315. 32. Joshi, V., Shivach, T., Yadav, N., and Rathore, A. S. (2014) Circular Dichroism Spectroscopy as a Tool for Monitoring Aggregation in Monoclonal Antibody Therapeutics. Anal. Chem. 86, 11606–11613. 33. Froehlich, E., Mandeville, J. S., Jennings, C. J., Sedaghat-Herati, R., and Tajmir-Riahi, H. A. (2009) Dendrimers Bind Human Serum Albumin. J.

Phys. Chem. B 113, 6986–6993. 34. Nobbmann, U., Connah, M., Fish, B., Varley, P., Gee, C., Mulot, S., Chen, J., Zhou, L., Lu, Y., Sheng, F., Yi, J., and Harding, S. E. (2007) Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol. Genet. Eng.

Rev. 24, 117–128. 35. Narayanan, J., and Liu, X. Y. (2003) Protein Interactions in Undersaturated and Supersaturated Solutions: A Study Using Light and X-Ray Scattering. Biophys. J. 84, 523–532. 36. Berkowitz, S. A., Engen, J. R., Mazzeo, J. R., and Jones, G. B. (2012) Analytical

tools

for

characterizing

biopharmaceuticals

and

the

implications for biosimilars. Nat. Rev. Drug Discov. 11, 527–540. 37. Berkowitz, S. A., Krull, I., and Rathore, A. (2015) Challenges in the Determination of Protein Aggregates, Part I. LCGC North America 33, 42-49. 31 ACS Paragon Plus Environment


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38.

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Kalhapure, R. S., and Akamanchi, K. G. (2012) Oleic acid based heterolipid

synthesis,

characterization

and

application

in

self-

microemulsifying drug delivery system. Int. J. Pharm. 425, 9–18. 39. Rubinstein, M., and Colby, R. H. (2003) Polymer Physics. New York:

Oxford University Press.


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Peptide Dendrons as Thermal-Stability Amplifiers for Immunoglobulin

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