Assessing the impact of formulation on protein-based biotherapeutics

Bradley, Derek1; Stocca, Alessia2; Burke, Mary1; Roche, James J.2

1 BioClin Research Laboratories, IDA Technology Park, Garrycastle, Athlone, Co. Westmeath, Ireland 2 Biosciences Research Institute, Athlone Institute of Technology, Dublin Road, Athlone, Co Westmeath, Ireland.

Abstract

Biopharmaceuticals are a class of revolutionary drugs that have been developed to treat a diverse range of serious

and often life-threatening diseases, such as cancer. As with small molecule drugs, they must be tested to ensure they

meet predefined specifications prior to release. However, owing to their complexity, the analytical challenges for

biopharmaceuticals are far greater than for small molecule drugs. Being protein in nature, biopharmaceutical

structure can be dramatically altered by formulation excipients. This may lead to an inefficacious drug, or indeed,

one which may have a significantly altered safety profile.

In this research, the following range of analytical techniques have been applied to two assumed-identical

asparaginase formulations, a biopharmaceutical that is used to treat acute lymphoblastic leukaemia (ALL) in children:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Intact mass (‘top-down’) analysis – mass spectrometry

Peptide mass fingerprinting (primary structure analysis)

Amino acid compositional analysis

Spectroscopic analysis – circular dichroism (CD)

Results show that the formulations exhibit highly similar responses in some analytical techniques, but not others.

This demonstrates that in order to characterise the impact of formulation on biological medicines, an orthogonal

analytical approach, comprising a battery of techniques which offer complimentary information, should be

employed.

Introduction

Biopharmaceuticals are protein-based therapeutics, which only function as intended when they adopt the correct

structure and three-dimensional shape. Proteins have several layers of assembly, each of which play a critical role in

determining overall protein shape. Alterations at any level of structure may result in an incorrectly folded protein,

which could give rise to a molecule with reduced efficacy, or potentially trigger a harmful immune response.(1-2)

Formulation of a biopharmaceutical product can dramatically influence the stability and structure of the protein, due

to interactions between the amino acid variable groups and formulation excipients or processes.(3) For example,

development of modified forms (such as aggregates) may be promoted during lyophilisation,(4) while certain

excipients may contribute to chemical modifications, such as oxidation.(5) Therefore, a key aspect of product

development is that the influence of formulation on stability and structure of biologics should be thoroughly

characterised.

This research is a joint industry/academia collaborative effort which aims to explore and develop methods to

characterise biopharmaceuticals. Development of robust techniques capable of reliably detecting differences in

biopharmaceutical structure is critical to ensuring patient safety. The rapid emergence of biosimilars (copy-versions

of innovator biologics authorised under abbreviated licensure pathways) underlines the increasing need for

availability of such techniques.

Methods and Materials

Samples for all tests were lyophilisates which were reconstituted in water according to manufacturer’s instructions –

final concentration was approximately 13.0 mg/mL. Reconstituted material was stored frozen at -80ºC in single-use

aliquots. When required, aliquots were thawed at room temperature. The following sections briefly describe the

methods for each test (experimental settings were optimised for each test during pilot-scale experiments) – further

details can be obtained by contacting the author.

SDS-PAGE: Samples were diluted with water to 1.0, 0.5 and 0.2 mg/mL. 100 µL of each dilution was combined with

an equal volume of 2X Laemmli sample buffer containing 100 mM DTT – this gives final protein concentrations of

0.5, 0.25 and 0.1 mg/mL, respectively. 20 µL of each preparation was pipetted into the wells of an ‘Any-kD, Stain-

free’ polyacrylamide gel (BioRad, product code # 456-8123), according to Figure 1 – this volume corresponds to a

total protein load of 10, 5 and 2 µg, respectively. Samples were electrophoresed at 200 V (constant voltage) using

the Mini-Protean electrophoresis system from BioRad – run time was 30 minutes. The gel was then imaged using the

‘Stain-free’-enabled ChemiDoc MP Imaging system from BioRad. Data was processed using ImageLab Software

version 5.4.1., Security Edition.

Intact mass analysis: Samples were diluted in 50% (v/v) acetonitrile containing 0.1% (v/v) formic acid to a final

concentration of 50 µg/mL. Samples were then infused using a Harvard 22 apparatus and syringe with a flow rate of

20 µL/min into the electrospray ionisation source of the API3000 mass spectrometer (ABSciex). The mass

spectrometer ionisation and detection settings were as detailed in Table 1, below. Data was analysed using Analyst

version 1.4 software.

Table 1: Instrument settings for intact mass analysis via MS

Peptide mass fingerprinting: Samples were diluted to 1.0 mg/mL with water. 50 µL of each sample (corresponding

to 50 µg of protein) was desalted using a solid phase extraction cartridge containing C8 resin. Samples were

evaporated to dryness in a vacuum centrifuge, then resuspended in 20 µL of 50 mM ammonium bicarbonate

containing 8 M urea. 5 µL of 45 mM dithiothreitol (DTT) was added followed by incubation at 60ºC for 45 minutes,

during which disulfide bonds were reduced. Then, 10 µL of iodoacetamide (IAA) was added, and tubes incubated at

room temperature in the dark for 45 minutes, during which free cysteines were alkylated to prevent disulfide bonds

reforming. A further 5 µL of 45 mM DTT was then added to neutralise excess IAA. Final volume was brought to 200

µL with water in order to dilute urea to below 1.0 M, as high urea concentration is not compatible with trypsin. 2 µL

of 1.0 mg/mL sequencing-grade trypsin was then added, and tubes incubated at 37ºC for 24 hours. Following

incubation, digestion was stopped by freezing the samples at -20ºC until required for analysis. Samples were diluted

1:1 with mobile phase A (see below) and 40 µL injected. Analysis was on the API3000 LC-MS/MS (ABSciex) system

with Perkin Elmer 200 Series autosampler and quaternary pump. Mass spectrometer settings were as detailed in

Table 1, with the exception that source temperature was 400ºC. The column was a C18-AR type, with dimensions of

4.0 mm (I.D.) X 150 mm (length) with 3 µm particle size. Samples were eluted with a 120 minute linear gradient from

2% (v/v) acetonitrile with 0.1% (v/v) formic (Mobile phase A) acid to 50% (v/v) acetonitrile with 0.1% (v/v) formic

acid (Mobile phase B). Flow rate was 1.0 mL/min, which was split with approximately 0.5 mL/min going to the source

(the remainder going to waste). Data was collected and analysed using Analyst version 1.4 software.

Amino acid compositional analysis: Samples were prepared at a concentration of 1.0 mg/mL. 2 µL of each sample (2

µg of protein) was added to an autosampler vial and evaporated to dryness. 200 µL of 6 N HCl containing 0.1% (w/v)

phenol was added, then the headspace of tubes purged with a stream of nitrogen before being capped tightly. Tubes

were then placed in a vacuum oven at 115ºC for 24 hours, during which peptide bonds are hydrolysed, releasing the

free amino acids. Following hydrolysis, HCl was evaporated off, then amino acids derivatised using the Waters AccQ-

Tag chemistry package. Analysis was on the Varian ProStar Fluorescence HPLC System. The column was a Nova-Pak

C18, (4.0 mm I.D., 150 mm in length, 4 µm particle size). A linear gradient from 2% to 50% B over 60 minutes, with a

flow rate of 1.0 mL/min was used for analysis. Injection volume was 5 µL. Standards from 0 to 100 µM of each amino

acid were also prepared from commercially available amino acid standards. These were also derivatised and

analysed in order to quantify the amount of each respective amino acid present in the test samples. Data collection

and processing was performed using Varian Star Chromatography Workstation software.

Circular dichroism: CD analysis was performed at Applied Photophysics, Leatherhead, UK. Samples were diluted

approximately 1:20 with water to reduce absorbance to about 1.8 A.U., which is required for CD analysis. A

lyophilised vial of asparaginase was also analysed – this was also diluted with water in order to obtain an absorbance

of approximately 1.8 A.U. Samples were scanned on the Chirascan Plus qCD Spectrometer. Analysis was performed

in isothermal mode (at 20ºC) in the far UV region (260 to 180 nm), with a wavelength increment of 1 nm, and a

bandwidth of 1 nm. Samples were also analysed with temperature ramping in order to compare melting

temperatures. For temperature ramping experiments, the scan range was from 250 to 190 nm, wavelength

increment of 1 nm, bandwidth of 1 nm, and temperature ramped from 20ºC to 90ºC in 1ºC increments. Applied

Photophysics’ Global 3 software was used for data analysis.

Results

Results from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Figure 1: Left – gel imaged following electrophoresis with bands for standards and samples auto-detected in ImageLab

software v. 5.4.1. Right – green filter applied to image revealing the presence of additional high molecular weight protein bands (red arrows)

Table 2: Molecular masses estimated from SDS-PAGE

Results from intact mass analysis via mass spectrometry

Figure 2: Centroided spectra from electrospray ionisation mass spectrometry of asparaginase samples: Left – M030614; Right – R030614.

Figure 3: Characteristic electrospray ionisation charge envelope for M030614 in profile; right – calculation of intact molecular mass from charge envelope data.

Results from peptide mass fingerprinting via ESI LC-MS

Figure 4: Peptide mass fingerprints: Top – recombinant asparaginase; Bottom – non-recombinant asparaginase.

In each figure, the top panel represents the total ion current (TIC) for peptides. The three lower panels are extracted ion chromatograms

for three selected peaks, which gives the molecular mass of the peptide fragments, which allows their identity to be confirmed

Results from amino acid compositional analysis

Figure 5: example standard curve for isoleucine showing excellent correlation

Figure 6: Representative chromatogram for 75 µM derivatised amino acid standard preparation

Table 3: Comparison of the amino acid composition of asparaginase samples

Table 4: Correlation of amino acid standard curves from amino acid analysis

Results from circular dichroism analysis

Discussion

Intact mass analysis via mass spectrometry revealed that the active ingredient in both preparations had the same

molecular mass (to within 1 Da of each other, which is below the 10 p.p.m. error typically allowed when reporting

intact mass results for proteins) (Figures 2 and 3). Post-translation modifications such as glycosylation, or sample

degradation processes, such as oxidation or deamidation, would have a major impact on intact molecular mass.

However, this was not observed for these samples, which indicates that the samples had not undergone such

changes. Peptide mapping data provided confirmation that the amino acid sequence of each protein sample was the

same, as digestion with the same proteolytic enzyme (trypsin) under the same experimental conditions yielded

identical fragments (Figure 4).

These data support the assumption that the active ingredient (asparaginase) in both preparations was identical.

However, results from circular dichroism analysis and SDS-PAGE suggest that differences exist between the

formulations. Very significant differences in CD spectra from 200 to 180 nm were observed (Figures 7 to 9). This

could suggest a secondary or tertiary structural change (i.e. how the protein is folded in three-dimensional space),

but could also be explained by the presence of far U.V-absorbing excipients in the recombinant formulation, that are

not present in the non-recombinant version. This could be further explored by repeating the CD analysis, but this

time, removing potential excipients via solid-phase extraction prior to analysis.

Analysis via SDS-PAGE revealed that there were some additional high-molecular mass bands in the non-recombinant

asparaginase (with molecular masses ranging from 50 to 100 kDa, by reference to the molecular mass standards)

(Figure 1). These may potentially be aggregates of asparaginase (dimers of the asparaginase protein unit). The

presence of these aggregates in one formulation but not in the other is potentially due to the differences in

formulation that are evident from CD analysis, as some excipients may prevent (or promote) aggregate formation,

particularly during storage.

Results from amino acid compositional analysis compared quite well between both asparaginase samples, with 14

out of 17 amino acids having relative standard deviations of less than 15%. However, experimental results did not

correlate with the expected composition. This is thought to be due to trace levels of some amino acids, such as

glycine, in the reagent-grade chemicals used. Subsequent work on amino acid analysis will attempt to eliminate

these sources of contamination. Standard curves for each amino acid have correlation coefficients of > 0.99.

Conclusions

The impact of formulation excipients on the structural characteristics of biopharmaceuticals should be thoroughly

evaluated. A battery orthogonal analytical approaches should be employed, as differences (such as propensity to

form aggregates which may impact on product safety or efficacy), may not be readily revealed using techniques such

as ‘top-down’ mass spectrometry alone.

References

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predictors of aggregation propensity. Computers & Chemical Engineering. 2013 Nov 11;58:369-77.

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Contact Information

Mary Burke

Managing Director,

Biopharmaceuticals Department,

BioClin Research Laboratories,

IDA Business & Technology Park

Garrycastle, Athlone,

Co. Westmeath

N37 X061

Email: jroche@ait.ie

Phone: +353 (0) 90 6468087

Fax: +353 (0) 90 646 8148

Derek Bradley (M.Sc.)

Senior Scientist

Biopharmaceuticals Department

BioClin Research Laboratories,

IDA Business & Technology Park

Garrycastle, Athlone,

Co. Westmeath

N37 X061

Email: derek.bradley@bioclinlabs.com

Phone: +353 (0) 90 646 0200

Fax: +353 (0) 90 646 0210

Acknowledgements

We wish to thank Applied Photophysics, Leatherhead, UK for performing

circular dichroism analysis and providing the data included on this poster.