Impact of Excipient Extraction and Buffer Exchange on Recombinant Monoclonal Antibody Stability

Abstract

The foundation of a biosimilar manufacturer’s regulatory filing is the demonstration of analytical and functional similarity between the biosimilar product and the pertinent originator product. The excipients in the formulation may interfere with characterization using typical analytical and functional techniques during this biosimilarity exercise. Consequently, the producers of biosimilar products resort to buffer exchange to isolate the biotherapeutic protein from the drug product formulation. However, the impact that this isolation has on the product stability is not completely known. This study aims to elucidate the extent to which mAb isolation via ultrafiltration-diafiltration-based buffer exchange impacts mAb stability. It has been demonstrated that repeated extraction cycles do result in significant changes in higher-order structure (red-shift of 5.0 nm in fluorescence maxima of buffer exchanged samples) of the mAb and also an increase in formation of basic variants from 19.1 to 26.7% and from 32.3 to 36.9% in extracted innovator and biosimilar Tmab samples, respectively. It was also observed that under certain conditions of tertiary structure disruptions, Tmab could be restabilized depending on formulation composition. Thus, mAb isolation through extraction with buffer exchange impacts the product stability. Based on the observations reported in this paper, we recommend that biosimilar manufacturers take into consideration these effects of excipients on protein stability when performing biosimilarity assessments.

1. Introduction

Biopharmaceutical products have revolutionized healthcare and have emerged as more effective (than the conventional small molecule-based pharmaceutical products) therapies for management of complex and otherwise difficult to manage diseases, including cancer, arthritis, and autoimmune disorders.¹ The active pharmaceutical ingredient (API) in a biologic formulation may be a (i) protein such as a monoclonal antibody (mAb), (ii) an attenuated form of a microbe, such as a vaccine, (iii) an oligonucleotide, or (iv) genes, cells, and tissues in the emerging advanced medicinal therapy products.²⁻⁴ Numerous protein therapeutic products have been approved, including monoclonal antibodies (mAbs), antibody-drug conjugates, bispecific antibodies, and fusion proteins.^5,6 Among these, mAb products are the most popular, evident from the number of approvals each year.⁷ However, the complex production process involved in mAb manufacturing poses affordability and accessibility challenges, especially in developing economies.⁸

Industry manufacturers and regulatory agencies worldwide strive to approval pathways for affordable biologics such as biosimilars.⁹⁻¹¹ Biosimilars are biologics that are highly similar to the original product (innovator) in terms of quality, safety, and efficacy.¹²⁻¹⁴ The requirements for demonstration of the similarity of biological products, from the viewpoints of safety and efficacy, are stringent. The guidelines from the World Health Organization are designed to serve as a framework for the evaluation of biosimilars across the globe.¹⁵ The regulatory agencies, for example the Food and Drug Administration and the European Medicines Agency, have their own guidelines as well.^4,16,17,18 Formulation development of biosimilars also entails careful and judicious excipient selection so as to provide a stable environment to the protein during both the product manufacture and its lifecycle. Establishment of analytical and clinical biosimilarity with the innovator product is a key step toward regulatory approval.^17,19 Generally, the analytical and functional biosimilarity assessment involves characterization of the amino acid sequence, higher-order structure (HOS), purity profile, aggregates, intact and reduced mass, potency, and binding kinetics of the biosimilar and the innovator product.^17,20 Direct analytical characterization of the mAb in a drug product (DP) is difficult because of interference from the formulation excipients.^22,23 Thus, in cases where excipient interference is involved, extraction of excipients is necessary from the formulated product before the API is characterized.²⁴ The biosimilarity assessment sometimes requires several processes such as buffer exchange to extract the API from formulated DP.^20,25,26 However, the impact of the processes used for extraction on the stability and functionality of the API is not known.²¹

Buffer exchange is a standard process in biotherapeutic manufacturing where the protein molecule is transferred from one buffer system to another through filtration and centrifugation processes.²⁷ During downstream purification (DSP), the purified protein product or drug substance (DS) is transferred from the chromatography elution buffer into the chosen formulation buffer to form the final DP. This is achieved through a combination of buffer exchange and concentration technology consisting of ultrafiltration–diafiltration (UF–DF) steps.²⁸ The UF–DF technology is highly efficient at low protein concentrations. However, when the protein concentration is high, it may lead to membrane fouling, interfacial stress, and cavitation, subjecting the protein to denaturation, precipitation, and unfolding.^29,30 Apart from manufacturing, buffer exchange is also a routine process in analytical testing to characterize mAb molecules without excipient interference. For example, during mass spectrometry (MS)-based characterization, such as peptide mapping and intact mass analysis, the excipient components can affect ionization and resolution,³¹ thereby necessitating removal of such components through the buffer exchange process. Similarly, circular dichroism (CD) is affected by mAb buffer components during analysis, making buffer exchange an obligation.³² While the removal of excipients through small-scale buffer exchange methods such as centrifugal concentrators can affect mAb stability and induce artifacts, these have not been investigated.

The current literature on the impact of buffer exchange extraction on stability focus on analyzing concentration effects of protein during UF–DF operations.²⁹ Specifically, the impact of shear stress and temperature on the aggregation of fusion proteins after UF–DF has been investigated. For example, in the UF process, shear stress at the protein solution and filter membrane interface causes protein–protein interactions and conformational changes, leading to aggregates and particle formation.³³ The high protein concentration may also cause membrane fouling due to polarization effects, ultimately leading to aggregation.³³ Lastly, the Gibbs–Donnan effect at different pH values and electrolyte concentrations in the UF–DF cycle can induce charge modifications in mAb, such as oxidation.³⁴ While the impact of buffer exchange during manufacture is discussed,³⁵ the impact of buffer exchange, if done prior to analytical characterization and comparability, on product stability has not received much attention.

Ion-exchange chromatography (IEC) usually forms the polishing final step in DSP purification, thereby making IEC elution and formulation buffers prominent in buffer exchange processes. The differences in strength, pH, and composition of the two buffer systems can cause protein instability due to the unfavorable conditions during buffer exchange.²⁹ Hence, the impact of the extraction process between elution and formulation buffers must be carefully assessed. To the best of our knowledge, there has so far been no comprehensive evaluation of the impact of buffer exchange, between elution and formulation buffers, on mAb stability if it is done prior to analytical and functional characterization. Thus, the present study aims to assess the effect of the buffer exchange process between the elution and formulation buffer on mAb stability. An innovator and its biosimilar product were subjected to alternate cycles of formulation and elution buffer. The objective is to understand the extent to which buffer exchange during manufacturing steps affects mAb stability. Additionally, excipient extraction achieved during the buffer exchange process further confirms the role of formulation excipients to restabilize protein structure and function. Lastly, the impact of excipient extraction on the mAb molecule related to size, charge, and HOS analysis has been tested.

2. Experimental Section

2.1. Materials

LC grade chemicals were used in the study. Sodium dihydrogen phosphate, sodium azide, isopropanol (>99.5%), polysorbate 20, sodium chloride (NaCl), orthophosphoric acid, formic acid (FA), and tris(hydroxymethyl) aminomethane were purchased from Merck (Rahway, USA); disodium hydrogen phosphate, l-histidine, l-histidine hydrochloride (HCl), trehalose dihydrate, and iodoacetamide (IAM) were purchased from Sigma-Aldrich (Missouri, USA); acetic acid (99%) was purchased from SRL Chemicals (Mumbai, India); sodium hydroxide was purchased from Himedia (PA, USA); dithiothreitol (DTT) was purchased from Promega Biotech (Madison, USA); MS grade trypsin was purchased from EDNA (New Delhi, India); and acetonitrile (>99.9%) of MS grade was purchased from JT Baker (Phillipsburg, USA). Quartz glass cuvette (1 mm), black polystyrene 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany), 0.22 μm membrane filter, centricons 30 kDa and Nanosep (30 kDa) (Pall Corporation), 96-well UV microplates (Corning, US), and recombinant human HER2 protein (R & D Systems) were also used. Water (MQ water) was collected from a Milli-Q water purification system Indion Lab-Q water maker (India). Trastuzumab (Tmab) innovator has been approved in United States as Herceptin and in Europe and India as Herclon, together with multiple biosimilars across these regions. Hence, it was chosen for the present excipient extraction study. One batch of the Indian innovator product (Herclon, Roche; 440 mg; batch Number N3970B04 B3536; expiry date: 02/2025) and one batch of a biosimilar formulated and manufactured in India (Eleftha, Intas Pharmaceuticals; 150 mg; batch number 26020038; expiry date: 11/2025) were acquired for the study. Eleftha is one of the approved Tmab biosimilar in India. In the present study, innovator Tmab refers to the Herclon product, and biosimilar Tmab refers to the Eleftha product.

2.2. Sample Preparation

Lyophilized innovator and biosimilar Tmab were weighed and reconstituted in the appropriate formulation buffers to obtain the desired protein concentration. The initial Tmab concentration in each commercial product was measured by ultraviolet (UV) spectroscopy before the extraction process. The initial Tmab concentration measured was 21.90 mg/mL for biosimilar Tmab and 22.89 mg/mL for innovator Tmab. The initial amount of protein for both control and extracted samples was precalculated as per requirement after each cycle for the set of analytical tools used, assuming a maximum of 20% protein loss after each cycle. All measurements were carried out in triplicate, and unless otherwise stated, the average value is reported.

IEC elution buffer composition was 50 mM sodium phosphate and 150 mM NaCl, pH 6. IEC elution buffer had no excipients. Formulation buffer composition was as in marketed DP (both innovator and biosimilar) with 0.30 mg/mL l-histidine, 0.47 mg/mL histidine HCl, 19.1 mg/mL trehalose dihydrate, and 0.09 mg/mL polysorbate 20 (pH 6).

2.3. Excipient Extraction and Buffer Exchange Procedure

The excipient extraction and buffer exchange were carried out using a 30 kDa centricon, with each cycle of around six wash volumes. Figure 1 illustrates the procedure of the buffer exchange and excipient extraction. For excipient extraction (Figure 1B), reconstituted Tmab is buffer exchanged from formulation buffer to IEC elution buffer and then exposed to repeated alternate buffer exchange cycles of formulation buffer and IEC elution buffer. Formulation buffer is with excipients, and the IEC elution buffer is without any excipients. IEC elution buffer was chosen since it is often the polishing step of purification before UF–DF buffer exchange from DS to DP.^36,37 Thus, when buffer exchange from formulation buffer into the IEC elution buffer is performed, Tmab is in a buffer without excipients, and excipient removal is achieved. Again, in the next cycle, Tmab is buffer exchanged from IEC elution buffer into the formulation buffer with excipients, and excipient addition is achieved. Hence, this repeated procedure of excipient addition and removal from Tmab through buffer exchange is termed the excipient extraction process. When repeated buffer exchange of Tmab is carried out only in formulation buffer (Figure 1A), it serves as the control of the experiment without any excipient addition or removal.

Figure 1.

Schematic representation of the excipient extraction and buffer exchange workflow. (A) Buffer exchange process only in formulation buffer (control). (B) Excipient extraction through buffer exchange in formulation followed by IEC elution buffer. Therefore, at the end of cycles 1, 3, 5, and 7 Tmab will be IEC elution buffer, and after cycles 2, 4, 6, and 8, Tmab will be in formulation buffer. In the IEC elution buffer cycles, there will be no excipients. The blue (left side) and the pink (right side) colors, respectively, reflect the presence and absence of the excipients.

IEC elution buffer is chosen as it forms the final step in DSP before the final formulation of Tmab. Tmab sample is aliquoted after each step of buffer exchange and excipient extraction cycle for stability analysis.

2.4. Methods

2.4.1. Size Exclusion Chromatography

Size-exclusion chromatography (SEC) was performed using Dionex Ultimate 3000 HPLC (Thermo Scientific, USA) on the TSKgel G3000SWxl Tosoh (7.8 × 300 mm) column from Tosoh, Tokyo. Chromatography was performed in the isocratic mode at a 0.50 mL/min flow rate with aqueous 50 mM phosphate buffer (pH 6.5), 300 mM sodium chloride, and 0.02% w/v sodium azide. The column temperature was maintained at 25 °C. All the buffers were filtered through a 0.22 μm filter. The samples were diluted in the same buffer, depending on UV-based concentrations after each cycle (dilution factor ∼4 if concentration ∼20 mg/mL), to yield a final concentration of 5 mg/mL for each sample, and 20 μg of protein was injected (in triplicates) into the column.

2.4.2. Weak Cation-Exchange Chromatography

Weak cation-exchange chromatography (WCX) was performed to quantify charge variants on a BioMab, NP5, PK column (4.6 mm × 250 mm, 5 μm, Agilent) operated at 25 °C using a Dionex Ultimate 3000 RSLC system (Thermo Scientific). Prior to injection, the column was saturated with 65% mobile phase A (15 mM sodium phosphate buffer and 0.05% NaN₃ at pH 6.2) and 35% mobile phase B (150 mM sodium phosphate buffer and 0.05% NaN₃ at pH 6.2). All buffers were filtered with 0.22 μm filters. Charged species were separated using a runtime of 23 min with a linear gradient from 35 to 65% B at a flow rate of 0.800 mL/min. The samples were diluted, depending on UV-based concentrations after each cycle (dilution factor ∼4 if concentration ∼20 mg/mL), to make final concentration of 5 mg/mL for each sample, and 30 μg of protein was injected (in triplicates) into the column.

2.4.3. Peptide Mapping

Samples (100 μg) were denatured using 8 M urea in tris buffer (0.4 M, pH 7.8). mAb was incubated with 5 μL of DTT (200 mM) for 1 h, followed by addition of 20 μL (200 mM) of IAA for 1 h. Furthermore, samples were diluted 10-fold with 50 mM tris pH 7.8 and digested using MS grade trypsin (1:20 w/w) with overnight incubation at 37 °C and acidified with FA. Peptides obtained by trypsin digestion were injected on the AdvanceBio peptide mapping C18 column (2.1 × 150 mm, 1.7 μm, Agilent Technologies) using a 1290 Infinity UHPLC system coupled with AdvaceBio 6545XT Q-TOF system (Agilent Technologies). The column was operated at a flow rate of 0.50 mL/min at 55 °C. Mobile phase A consisted of 0.1% v/v FA in water, and mobile phase B was 0.1% FA in acetonitrile. The peptide was eluted by a linear gradient of mobile phase B (5–40%) for 30 min. The MS parameters were drying gas at 300 °C, drying gas flow of 11 L/min, nebulizer pressure of 35 psig, sheath gas temperature of 275 °C, sheath gas flow of 10 L/min, nozzle voltage of 4000 V, and fragmentor voltage of 175 V. MS–MS data were collected in the profile mode at a rate of three spectra per second with a 50–1700 m/z range. Data were acquired using MassHunter data acquisition software (Agilent Technologies). Data analysis was performed with BioConfirm software version 10.0 (Agilent Technologies).

2.4.4. CD

The far-UV CD experiment was performed using a Jasco-1500 spectrometer equipped with a Peltier temperature controller at 20 °C. Quartz glass cuvette with an optical path length of 0.10 cm was used, and analyte at 0.3 mg/mL concentration (diluted in Milli-Q from 5 mg/mL of near-UV CD sample with dilution factor 16.67) was scanned in the wavelength range of 190–250 nm. Corresponding extraction buffer was used as a blank. The spectra obtained were baseline corrected by using device software. Similarly, the near-UV CD experiment was performed on each sample diluted to 5 mg/mL in Milli-Q (dilution factor ∼4 if concentration ∼20 mg/mL) over a wavelength range of 250–350 nm.

2.4.5. Fourier Transform Infrared Spectroscopy

The measurements were performed using a Nicolet iS50 Fourier transform infrared (FTIR) spectrometer from Thermo Scientific, USA running on Omnics software. Each sample was diluted to 5 mg/mL (dilution factor ∼4 if concentration ∼20 mg/mL), and 6–8 μL of the sample was loaded. The corresponding extraction buffer was measured as a blank.

2.4.6. Fluorescence Spectroscopy

Fluorescence (FLR) measurements were performed using a Cary Eclipse fluorescence spectrophotometer by Agilent Technologies, Santa Clara, USA. In each well, 200 μL of the sample with protein concentration of 0.3 mg/mL (dilution factor ∼66.67 if concentration ∼20 mg/mL) was analyzed using black polystyrene 96-well microtiter plates (Greiner Bio-One GmbH, Germany). The spectrum for intrinsic fluorescence was recorded at 25 °C, from 300 to 450 nm with step size of 1 nm and at an excitation wavelength of 280 nm.

2.4.7. Ultraviolet Spectroscopy

Ultraviolet spectroscopy (UV) measurements were performed using Epoch microplate reader BioTek Instruments running Gen 5 version 1.11 software. Each sample was diluted (dilution factor 80) and loaded on to Corning UV plates. Corresponding extraction buffer was used as a blank, and the absorbance obtained was used to determine the concentration through reported molar extinction coefficient values of 1.435 mL/mg.cm obtained theoretically through the literature.³⁵ Since the concentration (mg/mL) provides the mass of protein (mg), it enabled the establishment of mass balance after each cycle.

2.4.8. Surface Plasmon Resonance

For surface plasmon resonance (SPR), recombinant human HER2 protein was immobilized on the surface of a CM5 chip by the amine coupling method (300 RU). Samples were diluted (dilution factor ∼20 if concentration ∼20 mg/mL) and injected at a series of concentrations ranging from 6.25 to 100 μM with association time 60 s followed by 90 s dissociation phase. All measurements were performed at 25 °C with a flow rate of 30 μL/min using HBS-EP buffer, as per the manufacturer’s protocol. Kinetic constants were calculated from the sensorgrams using the 1:1 fit model using BIA Evaluation 2.0.1 (Cytiva) software.

3. Results

In the description below, the control refers to the sample in which the repeated buffer exchange steps were followed only in the formulation buffer for both the innovator and biosimilar Tmab. Extracted samples refer to the systems in which the excipient extraction through buffer exchange was performed against IEC elution buffer for the innovator and biosimilar Tmab (as described in Section 2.3).

3.1. High-Molecular-Weight Impurities

Neither dimer formation nor other high-molecular-weight (HMW) impurities were observed in both control and extracted samples by the end of the last cycle (Supporting Information, Figure S1). Thus, with respect to HMW formation, addition or removal of excipients and the buffer exchange steps did not impact Tmab stability in both the biosimilar and innovator products.

3.2. Charge Variants

During the buffer exchange and excipient extraction cycles, Tmab was not subjected to any pH or temperature changes, to ensure that the sole stress is due to excipient extraction or through repeated buffer exchange. Figure 2 summarizes the trend of charge variants in the innovator and biosimilar Tmab samples during the buffer exchange and excipient extraction processes. The charge variants for control samples (both innovator and biosimilar Tmab) are essentially unchanged from the start until the end of the last buffer exchange cycle, indicating that the buffer exchange does not impact the charge heterogeneity in Tmab samples.

Figure 2.

Graphical representation of charge variants in innovator and biosimilar Tmab samples; (A) acidic variants in control and extracted Tmab samples; (B) basic variants in control and extracted Tmab samples. The buffer exchange cycle proceeds for up to 10 cycles of buffer exchange, whereas the excipient extraction proceeds only for up to 8 cycles due to higher protein loss (Section 3.5 of mass balance).

In contrast, the acidic variants in the extracted innovator Tmab samples gradually decrease as we reach the end of the last cycle, whereas an abrupt drop in acidic variants (from 17.19% after cycle 6 to 13.12% after cycle 8) during excipient extraction is witnessed for the extracted biosimilar Tmab sample (Figure 2A). Also, the basic variants gradually increase from 19.07 to 26.65% in extracted innovator Tmab and from 32.33 to 36.87% in extracted biosimilar Tmab samples (Figure 2B). The acidic variants are affected by deamidation in mAbs, and the gradual decrease in acidic variants of the extracted innovator Tmab sample shows that the stability of innovator Tmab gradually decreases (Figure 2A), whereas in the case of the extracted biosimilar Tmab sample, the acidic charge heterogeneity is stable up to cycle 6 and then abruptly decreases after cycle 7. The different buffer conditions encountered by mAbs during buffer exchange in manufacturing are often stabilized by the formulation buffer.³⁰ Here, we expose the Tmab to repeated extraction (between the IEC elution buffer and formulation buffer) for up to eight cycles. Though stabilizing effects of formulation buffer are witnessed until cycle 6 for biosimilar Tmab, the repeated extraction process ultimately leads to decrease in acidic variants. Furthermore, slightly acidic pH of formulation buffer is known to cause isomerization in mAbs³⁹ that accumulates with extraction stress, gradually for innovator Tmab and after cycle 6 for biosimilar Tmab. Thus, in the case of innovator Tmab, the stabilizing effect from excipients is not enough to control variations in acidic impurities from the first cycle itself, whereas the stabilizing effect of excipients in biosimilar Tmab loses effect after cycle 6. Impact of variations in shear stress, interfacial stress, isomerization, and ionic strength variations surpass the stabilizing effect of excipients, earlier for innovator Tmab and after cycle 6 for biosimilar Tmab. Overall, the excipient extraction in Tmab impacts both the acidic and basic variants.

Several factors such as pH and temperature influence the Asp isomerization in Tmab, including the low pH of Tmab formulation buffer. Usually, the pH of mAb formulations is slightly acidic, which prevents the formation of aggregates and reactions like oxidation and deamidation but cannot prevent Asp isomerization.³⁹ In our study, IEC elution buffer and formulation buffer were kept at a constant pH of 6 without any temperature changes, and hence, changes encountered are only due to excipient extraction. Since isomerization does not induce a mass difference, it is detected only through retention time difference (RT) of the eluted peaks in IEC (Supporting Information, Figure S2) and peptide mapping analysis.³⁹⁻⁴¹ The decrease in acidic variants of about 1.65–4.21% and increase in basic variants of 4.34–7.58% for innovator and biosimilar Tmab samples could be due to deamidation conversion to isomerization and intermediate succinimide modifications. The discussion section further elaborates on the isomerization and peptide mapping data based on the literature and elution profiles.

3.3. Tertiary Structure

The tertiary structure of a protein is depicted through the changes in absorbance of aromatic amino acid residues during near-UV CD and FLR analysis.⁴² Near-UV CD spectra were taken between 250 and 350 nm to obtain the absorbance of three primary aromatic acid residues: tryptophan (W) at 297 and 284 nm, tyrosine (Y) at 277 nm, and phenylalanine (F) at 268 and 261 nm.³⁸ Alterations in mdeg intensities at specified wavelengths corresponding to respective amino acids reflect disruptions caused by unfolding or aggregation in a mAb molecule.⁴³Figure 3 represents the near-UV CD spectra for the control and extracted samples. Graphical data for the same have been represented in the Supporting Information, Figure S3. For control biosimilar Tmab samples (Figure 3A), spectral intensities at positive 290 nm show variations from run to run but are stabilized by the end of the last cycle. Changes in control innovator Tmab samples follow similar variations but are more pronounced with an upward shift in tryptophan intensities (Figure 3B). Similar results are observed for the graphical plot of tryptophan intensities (Supporting Information, Figure S3). Though tyrosine intensities of both innovator and biosimilar Tmab were disrupted during buffer exchange (Supporting Information, Figure S3-A), they were stabilized by the end with the last cycle showing similar intensities to the first cycle. In contrast, phenylalanine intensities for control samples appeared to be stable throughout the buffer exchange cycle, suggesting that buffer exchange impacts mostly the tryptophan amino acid residues.

Figure 3.

Spectral representation of tertiary structures of extracted and control Tmab samples through near-UV CD analysis; (A): control biosimilar Tmab samples; (B): control innovator Tmab samples; (C) extracted biosimilar Tmab sample; (D) and extracted innovator Tmab sample. In all panels, E1–E8 represent cycle numbers for biosimilar Tmab, and H1–H8 represent cycle numbers for innovator Tmab. In panels C and D (for extracted samples), in cycles 1, 3, 5, and 7, Tmab is dissolved in IEC elution buffer (no excipients), while in cycles 2, 4, 6, and 8, the Tmab is dissolved in formulation composition (excipients + buffer).

For the extracted Tmab samples, variations in tryptophan spectral intensities were much more pronounced than those of control samples for both innovator and biosimilar Tmab samples (Figure 3C,D). Similar results are observed for the graphical plot of tryptophan intensities (Supporting Information, Figure S3-A). Thus, tryptophan residues continue to be affected by buffer exchange and excipient extraction. In contrast to the control samples, tyrosine spectral intensities depicted changes in the extracted samples (Figure 3B,C), also seen as a continuous increase in the graphical intensity plot of tyrosine (Supporting Information, Figure S3-B), suggesting excipient extraction impacts the tertiary structure of the Tmab. Lastly, the phenylalanine intensities are least affected by the extraction cycles and appear to be stable throughout. The spectral variations are further confirmed by graphical plots of near-UV CD data (Supporting Information, Figure S3).

Nature of the environment around aromatic residues (polar or nonpolar) and hydrogen bonding greatly influences the near-UV CD spectra.⁴⁴ Generally, tryptophan residues have a larger, more hydrophobic side chain, and relatively higher solvent accessibility than tyrosine and phenylalanine with more compact side chains.⁴⁵ Though solvent exposure is also dependent on amino acid residue location, tryptophan residues are more surface-exposed compared to the tyrosine and phenylalanine residues.⁴⁶ Consequently, major variations in tryptophan intensities are observed even during buffer exchange when tyrosine and phenylalanine residues are deeply buried and remain unaltered. When excipient extraction occurs, the differences in ionic strength and composition of buffers coupled with the absence of formulation stabilizers lead to enhanced disruptions in tryptophan residues. Additionally, changes in the buffer environment also impact the tyrosine residues, explained in the Discussion section, leading to disruptions in the near-UV CD spectra during excipient extraction. Lastly, phenylalanine remains the least affected by buffer exchange or excipient extraction. Though the extent of surface exposure of these amino acids is dependent on mAb unfolding under stress and needs further investigation, fluorescence intensities can give additional insights into the tertiary structure changes.

A decrease in spectral intensity and shift to longer wavelengths (red-shift) during the FLR analysis indicates exposure of the buried amino acid residues due to conformational changes and environmental impact on the protein structure. An increase in spectral intensity and shift to shorter wavelengths (blue-shift) associated with the increased hydrophobicity of the environment around the fluorophore when amino acids are buried inside the protein core.⁴²Figure 4 represents the red-shift and blue-shift observed for the control and extracted samples, and spectral intensity plots for the FLR analysis have been represented in Supporting Information, Figure S5. For control Tmab samples, stability in wavelength shifts is maintained until cycle 3 of buffer exchange, following which a red-shift was observed in both control innovator and biosimilar Tmab samples (Figure 4). By the end of buffer exchange cycle, the control biosimilar Tmab had a red-shift of 5.0 nm, whereas the control innovator Tmab showed a blue-shift of 1.0 nm. The spectral intensity plots for control samples (Supporting Information, Figure S5-A,B) also represent similar differences, wherein a red-shift is accompanied by a decrease in spectral intensity and a blue-shift by an increase in spectral intensity. Furthermore, the differences in spectral plots of control biosimilar samples between cycles are much wider than the control innovator samples, suggesting that buffer exchange impacts biosimilar Tmab more than the innovator Tmab.

Figure 4.

Graphical representation of the tertiary structure of control and extracted Tmab samples through FLR analysis. In cycles 1, 3, 5, and 7, Tmab is dissolved in IEC elution buffer (no excipients), while in cycles 2, 4, 6, and 8, the Tmab is dissolved in formulation composition (excipients + buffer). The buffer exchange cycle proceeds up to 10 cycles of buffer exchange, whereas the excipient extraction proceeds only up to 8 cycles due to higher protein loss (Section 3.5 of mass balance).

The extracted biosimilar Tmab sample also displays similar changes to control biosimilar, with a continuous and more pronounced red-shift after cycle 3 of extraction (Figure 4). The trend of wavelength shifts in extracted innovator Tmab samples is similar to that of the control innovator Tmab sample but appears more enhanced (Figure 4). The spectral plots (Supporting Information, Figure S5-B,C) reflect similar observations (red-shift is accompanied by a decrease in spectral intensity and a blue-shift by an increase in spectral intensity), especially for extracted innovator Tmab where differences in spectral intensity between extraction cycles is now more prominent. Thus, the tertiary structure of the control and extracted Tmab sample is disrupted by both buffer exchange and excipient extraction processes. Like charge variant analysis, where disruptions were observed primarily after cycle 6 in extracted biosimilar Tmab, changes to spectral intensity in fluorescence analysis are observed earlier after cycle 3 for all samples. It is speculated that the stabilizers in formulation buffer can suppress charge heterogeneity up to cycle 6 of extraction, but tertiary structure changes are not stabilized following cycle 3.

3.4. Secondary Structure

The far-UV CD changes in the secondary structure are reflected by millidegree (mdeg) intensities of beta structures that are characterized by a positive band at 202 nm and a negative band around 218 nm. A loss in the secondary structure is evident from the change in mdeg values at 202 and 218 nm.^38,47 No change in 202 and 218 nm intensities was observed for control innovator Tmab samples (Figure 5B), whereas the control biosimilar Tmab samples exhibited stability at 218 nm but were unstable at 202 nm at cycle 10 (Figure 5A). For extracted Tmab samples, degradation was observed at both 202 and 218 nm, suggesting that excipient extraction enhances disruptions of secondary beta structures (Figure 5C,D).

Figure 5.

Spectral representation of the secondary structures of extracted and control Tmab samples through far-UV CD analysis; (A): control biosimilar Tmab samples; (B): control innovator Tmab samples; (C) extracted biosimilar Tmab sample; and (D) extracted innovator Tmab sample. In all panels, E1–E8 represent cycle numbers for biosimilar Tmab, and H1–H8 represent cycle numbers for innovator Tmab. In panels C and D (for extracted samples), in cycles 1, 3, 5, and 7, Tmab is dissolved in IEC elution buffer (no excipients), while in cycles 2, 4, 6, and 8, the Tmab is dissolved in formulation composition (excipients + buffer).

Secondary structure changes in FTIR analysis were monitored by changes in absorbance intensity around wavenumbers 1624–1693 cm^–1, assigned as amide I bands.⁴⁸ Specific secondary structure absorbance for FTIR analysis at around wavenumber 1638 cm^–1 represents beta structures in mAbs that tend to decrease when mAb is subjected to unfolding or degradations, with a corresponding increase in absorbance for wavenumbers 1648 and 1685 cm^–1.⁴⁹ The control innovator Tmab samples at specified wavenumbers remain stable throughout the buffer exchange cycles (Supporting Information, Figure S4), whereas the control biosimilar Tmab samples tend to follow this trend as the last cycle is reached. Similarly, extracted innovator and biosimilar Tmab samples also follow the trend, indicating beta secondary structure degradations due to repeated excipient addition/removal (Supporting Information, Figure S4).

3.5. Mass Balance

Protein loss during buffer exchange is often observed due to protein adsorption to the membrane.⁵⁰ Mass balance during excipient extraction was calculated based on UV measurements after each cycle. Protein loss was higher for extracted Tmab samples, with a maximum percent loss of around 30.9% in innovator Tmab and around 25.6% in biosimilar Tmab (data not shown). Protein loss in control samples was comparatively less with a maximum of 14.9% in innovator Tmab and around 9.3% in biosimilar Tmab (data not shown). Solution pH, conductivity, ionic strength, and composition are crucial factors that are known to affect mAb interactions with membranes during buffer exchange.³³ During excipient extraction, Tmab is subjected to buffers of altered ionic strength and composition (formulation buffer and IEC elution buffer), leading to different mAb interactions with the membrane. Such interactions are low in the buffer exchange of control samples with only formulation buffer. Thus, subjecting Tmab to alternate conditions of buffer strengths during extraction results in more protein loss. Innovator and biosimilar samples were subjected to similar mechanisms of extraction and buffer exchange; hence, the difference in protein loss is unexpected but could be attributed to conformational changes the Tmab undergoes during the process.

3.6. Binding Assay

SPR was used to compare the binding kinetics between the samples and Her2 protein. Aspartic acid isomerization is reported to impact mAb binding activity.^41,51 Since the charge variant analysis revealed an increase in basic species, especially in cycles 6–8, these were used for the SPR analysis. Table 1 lists the equilibrium dissociation constant (K_D) values used to evaluate and rank order strengths of biomolecular interactions for these samples. The K_D values obtained were similar between all the samples and within the instrument’s acceptable range, confirming that there is little difference among them and were observed to be of the same order of magnitude among the samples.

Table 1. Table Summarizing the K_D Values Obtained for Extracted Samples.

extracted sample	KD value (M)	% basic variants
biosimilar Tmab cycle 6	1.13 × 10–8	32.98
biosimilar Tmab cycle 7	7.96 × 10–8	36.69
biosimilar Tmab cycle 8	9.57 × 10–8	36.87
innovator Tmab cycle 6	3.85 × 10–8	22.19
innovator Tmab cycle 7	4.04 × 10–8	26.01
innovator Tmab cycle 8	4.28 × 10–8	26.65

4. Discussion

The excipient extraction and buffer exchange cycles for innovator and biosimilar Tmab reveal crucial information about Tmab stability. Charge heterogeneity, binding affinity, and HOS assessment of the Tmab were performed using a range of analytical tools including IEC, SPR, SEC, FTIR, CD, and FLR.

Based on literature, aggregation after UF–DF buffer exchange is reported only after long-term storage of protein solution at elevated temperatures.²⁹ Our results show that there was no HMW formation in Tmab after repeated extraction with or without excipients, which is expected since we are using formulation buffer and did not perform long-term storage studies (Supporting Information, Figure S1). Modifications like isomerization, succinimide formation, unfolding, and hydrolysis can be possible attributes of Tmab stability during excipient extraction.

Deamidation of the asparagine (N) amino acid residue is a common chemical modification in Tmab.⁵² The asparagine deamidation reaction is influenced by pH and temperature, forming either aspartic acid (Asp) or iso-aspartic acid (isoAsp).⁵³ Formation of Asp leads to an increase in acidic variants of a mAb due to corresponding pI shifts.⁵⁴ Similarly, the formation of isoAsp or isomerization can form either acidic or basic species.⁴⁰ Asp and isoAsp are interchangeable products preceded by a reaction intermediate, mAb succinimide, also eluting as basic species in an IEC analysis.⁵⁴ The peptide mapping data (Supporting Information, Tables S1–S4) reveal deamidation and isomerization hotspots in extracted innovator and biosimilar Tmab samples. The aspartic acid (D) residues preceding serine (S), glycine (G), and threonine (T) residues are the most susceptible to isomerization.³⁹ Literature reveals Tmab isomerization hotspots to be isoAsp at position 30 in the complementary determining region 1 (CDR1), Asp 55 in CDR2, and D108 in CDR3. Similarly, deamidation at Asp74, LNG in constant domain (CH2), and 384 position of the heavy chain are highly prone to succinimide formation.⁵⁴ After cycle 1 of extraction, 14 deamidation sites in biosimilar Tmab and 7 aspartic-acid isomerization hotspots are revealed (Supporting Information, Table S1). Two already exist as isomerized forms owing to the RT in peptide mapping analysis. Similarly, 13 deamidation sites and three aspartic-acid isomerization hotspots are revealed in innovator Tmab after cycle 1 of extraction (Supporting Information, Table S3). Peptide mapping of extracted Tmab samples after the last cycle shows seven isomerized sites in biosimilar Tmab (Supporting Information, Table S2) and around five in innovator Tmab (Supporting Information, Table S4) owing to RT differences in identical peptides of same masses. The peptide map of extracted Tmab samples in the last cycle (Supporting Information, Tables S2,S4) exhibited an increase in isomerization modifications after deamidation of Tmab. Thus, a decrease in acidic variants and a subsequent increase of basic variants witnessed at the end of excipient extraction for extracted Tmab samples (Figure 2) are due to deamidation conversion to isomerization and intermediate succinimide modifications.

The tertiary structure of proteins consists of weak hydrogen or ionic bonds between amino acid side chains, which are responsible for protein–protein interactions.⁵⁵ Aromatic amino acids such as W, Y, and F contribute to the intrinsic fluorescence of Tmab. In the present study, in both control and extracted Tmab samples, spectral intensity and wavelength shifts are affected by the buffer exchange process (Figure 4). A red-shift after cycle 3 of excipient extraction or buffer exchange is predominant in all samples, especially the biosimilar Tmab. The innovator Tmab undergoes a red-shift followed by a blue-shift at the end of the last cycle (Figure 4). The control biosimilar Tmab exhibits moderate stability between cycle 5 and cycle 8 after repeated buffer exchange but unfolding is apparent by the end of cycle 10. In the case of extracted biosimilar Tmab sample, this trend is not witnessed, and a continuous increase in red-shift, after cycle 4 is observed. Thus, excipient extraction escalates the unfolding of biosimilar Tmab compared to buffer exchange. Blue-shift of wavelength in mAbs is observed when the environment around a fluorophore (e.g., tryptophan) becomes more hydrophobic or less polar, resulting from that changes that lead the aromatic amino acids to be buried deeper inside the hydrophobic core.⁵⁶ The control innovator Tmab shows a slight blue-shift by the end of the buffer exchange cycle, which is more pronounced in the extracted innovator Tmab sample. Since, no increase in the HMW species is observed for Tmab samples, a blue-shift could be due to a change in the hydrophobic environment of the aromatic amino acid chain during extraction. A possible explanation could be that the during initial cycles of buffer exchange and excipient extraction, the innovator Tmab undergoes partial unfolding due to shear and interfacial stress, evident from the red-shift in FLR (Figure 4). As buffer exchange proceeds, the formulation stabilizers are trying to stabilize disrupted and exposed structures in the control innovator Tmab. The presence of alternate IEC buffer during extraction hampers this stabilization by formulation components in extracted innovator Tmab, and enhanced disruptions are observed. The spectral plots (Supporting Information, Figure S5) further authenticate the changes to the tertiary structure observed during intrinsic fluorescence. An interesting study on higher-order structural analysis of mAbs through near-UV CD reports that changes in the buffer environment around tyrosine residues in the CDR region, specifically in formulation buffer, can impact the tertiary structure of proteins and influence Asp isomerization.⁵⁷ In the present study, Asp isomerization and near-UV CD spectra shift for tyrosine residues, specifically in extracted Tmab samples, confirm the impact of excipient extraction on the Tmab tertiary structure (Figure 3). As described, changes in the tryptophan spectra observed in near-UV CD analysis also contribute to tertiary structure disruptions in both control and extracted Tmab samples (Figure 3). Thus, an overall degradation of the innovator and biosimilar Tmab tertiary structure has been observed due to changes in the environment around tryptophan and tyrosine residues, possibly leading to unfolding of the Tmab molecule.

Both FLR and near-UV CD analysis confirm that buffer exchange affects Tmab (innovator and biosimilar) stability that is amplified after excipient extraction (Figures 3 and 4). Near-UV CD analysis provides us with an overall spectral change related to tryptophan, tyrosine, and phenylalanine residues during buffer exchange and excipient extraction. Specific intensity changes after cycle 3 of extraction visible in FLR analysis cannot be distinguished in near-UV CD analysis. Certain stabilization effects observed in innovator Tmab by FLR spectral intensities for control and extracted innovator Tmab are observed for tyrosine residues in near-UV CD analysis for control Tmab samples. Similarly, individual changes to tryptophan, tyrosine, and phenylalanine residues described separately for control and extracted Tmab samples are not discussed through FLR analysis as explained by the near-UV CD analysis. Overall, both FLR and near-UV CD analyses are needed to understand complete changes to tertiary structures during buffer exchange and extraction.

The predominant secondary structures in Tmab consist of beta-sheets and beta-turns, followed by random coils and α helix.⁵⁸ Far-UV CD spectra for extracted Tmab samples show differences at 202 and 218 nm, directly linked to beta secondary structural elements when compared to those of the stable control Tmab samples (Figure 5). FTIR and far-UV CD analysis show that while the buffer exchange has minimal impact on the Tmab secondary structure, the excipient extraction process impacts the secondary structure spectra of innovator and biosimilar Tmab. Whether these differences affect product quality attributes or clinical performance is still unknown.

Interfacial stress and shear during buffer exchange procedures are often regarded as contributing factors to mAb unfolding.³⁰ Since no increase in the HMW species of control or extracted Tmab samples has been observed, disruptions to HOS could be due to hydrolysis or Tmab unfolding.^59,60 Furthermore, increased degradation of the Tmab secondary and tertiary structures is observed in the present study when Tmab is exposed to repeated excipient extraction cycles. Though disruptions to the tertiary structure of Tmab are enhanced compared to the secondary structure degradations, a stabilization in the tertiary structure is also observed for some cases (Figures 3 and 4).

5. Significance

To receive regulatory approval for biosimilar products, manufacturers need to demonstrate that their products are highly similar with no clinically meaningful difference from the original product to guarantee the safety, purity, and potency of the biosimilar products under consideration. A limitation in this assessment is the interference that the excipients in the formulation cause for most of the analytical and functional techniques that are employed.⁶¹ Consequently, biosimilarity assessment involves extraction of the biotherapeutic protein from the innovator’s DP formulation. In this manuscript, we aim to elucidate the role of excipient extraction in destabilization of the protein product. It is observed that indeed the excipient removal impacts charge variant profiles as well as the secondary and tertiary structures of the protein. Though aggregation did not occur, changes in charge heterogeneity and HOS spectral differences are observed. Whether these differences affect product quality attributes or clinical performance is still unknown. The experimental plan adopted in the current study considered multiple cycles of buffer exchange and excipient extraction, contrary to the single-step buffer exchange that is typically followed in therapeutic product manufacturing. However, a gradual increase of basic variants with a subsequent decrease in acidic variants for the extracted innovator Tmab was observed immediately after the first cycle of buffer exchange. Additionally, changes in the tertiary structure were observed for both control and extracted samples from the first cycle in near-UV CD and after cycle 3 in FLR analysis. Similarly, far-UV CD also depicts distortions in the secondary structure, especially extracted biosimilar Tmab, after the first cycle of excipient extraction. The penultimate step in manufacturing requires a single step of UF–DF to formulation, but the subsequent analytical and functional characterization include multiple steps of buffer exchange and excipient extraction. For example, HOS characterization through CD requires complete removal of formulation stabilizers before measurements;²⁵ MS-based analytical methods like peptide mapping and intact mass analysis require removing all excipients through multiple steps of buffer exchange before analysis.^62,63 Thus, the experiments adopted in the present study attempt to understand the impact of these multitude factors of isolation on mAb stability. Nevertheless, primary extraction experiments performed in this study also have certain limitations. For instance, different mAbs and their biosimilars might show distinct stability changes with respect to buffer exchange and excipient extraction. Also, each mAb has a different formulation with a varied type or composition of excipients and is expected to depict variations in stability.

6. Conclusions

The impact of buffer exchange and excipient extraction on Tmab stability was evaluated with HMW, charge, HOS, and binding affinity analyses. Our results indicate that buffer exchange impacts innovator and biosimilar Tmab stability but also restabilizes the Tmab after destabilization in some cases, while degradations observed during excipient extraction are associated with irreversible changes. Whether these differences affect product quality attributes or clinical performance is still unknown. Charge heterogeneity changes like a decrease in acidic species with a corresponding increase in basic species point toward the formation of isomerization and succinimide products. Tmab HOSs are also disrupted by buffer exchange and excipient extraction. While the tertiary structure is impacted by both buffer exchange and excipient extraction, the secondary structure disruptions are limited to excipient extraction. The results of the study focus primarily on Tmab product with specific formulation evaluated wherein innovator and biosimilar Tmab primarily display similar behavior of destabilization after repeated extraction cycles, except for a few cases where innovator Tmab shows restabilization. The study also highlights the effect of Tmab extraction during buffer exchange processes on antibody stability. Manufacturers should be mindful of product degradation during the extraction process and use appropriate analytical and functional characterization tools to assess this degradation and ensure that it is not significant enough to diminish the outcome of the comparability exercise.

Glossary

Abbreviations used

mAbs

monoclonal antibodies

DP

drug product

DS

drug substance

DS

drug substance

UF–DF

ultrafiltration–diafiltration

FTIR

Fourier transform infrared spectroscopy

HOS

higher-order structure

CD

circular dichroism

FLR

fluorescence spectroscopy

SPR

surface plasmon resonance

CDR

complementary determining region

MS

mass spectrometry

IEC

ion-exchange chromatography

SEC

size-exclusion chromatography

IAA

iodoacetamide

DTT

dithiothreitol

FA

formic acid

W

tryptophan

Y

tyrosine

F

phenylalanine

Tmab

trastuzumab monoclonal antibody

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.3c01157.

• Peptide mapping results of innovator and extracted Tmab; graphical representation of SEC aggregate analysis; WCX-integrated chromatography peaks of innovator extracted Tmab sample with charge variant distribution; graphical data of near-UV CD analysis; spectra summarizing secondary structures of Tmab at specific wavenumbers (cm^–1) from FTIR analysis; and fluorescence spectral intensity plots for Tmab samples (PDF)

The authors declare no competing financial interest.