Automated cGMP optical labeling of FDA-approved antibodies for human use

Abstract

Monoclonal antibodies are commonly used as targeted therapies for autoimmune, infectious and oncologic diseases. Here, we describe a simple protocol to optically label monoclonal antibodies for use as molecular imaging agents for clinical investigation. In preclinical settings, optical imaging has complemented the strengths of nuclear imaging while offering higher resolution and a safer method for antibody-ligand engagement. However, translation of optically labeled monoclonal antibodies to the clinic has been slow because antibodies require a manual process in current good manufacturing practices (cGMP)-compliant facilities; the cost barriers to establish the investigational new drug application by using the traditional contract research organization pathway (>US$1 million) exceed the resources of academic institutions and early-phase pharmaceutical companies. To address these challenges, we repurposed an existing radiolabeling cGMP method for optical labeling that uses an automated, self-contained synthesis module. This method depends on commercially available, single-use, cassette-based production, which simplifies the workflow and does not require a clean room facility. This automated production method reduces both the cost and time required to produce a clinical dose of near-IR fluorescently labeled monoclonal antibody–IRDye800CW, decreasing the development costs for pilot and initial batches by almost 90%, as well as the production time by 40% to 4 h plus quality control (~10 h total). Our cGMP manufacturing method can optically label any compatible monoclonal antibodies at any dedicated radiochemistry facility. We provide the detailed protocol for production of panitumumab–IRDye800CW and nivolumab–IRDye800CW under cGMP regulations, achieving excellent yield, optimal degree of labeling and high purity.

Introduction

Since 1986, >100 monoclonal antibodies have been approved by the US Food and Drug Administration (FDA)¹, averaging approximately three approvals per year. Monoclonal antibodies have become the fastest-growing class of drugs to treat diseases^2,3 such as cancer, autoimmune disorders and chronic inflammatory diseases. Because of their high antigen specificity and low toxicity, they are used to selectively block receptor signaling, bind circulating molecules, deliver a therapeutic payload or serve as a diagnostic agent⁴. The use of optically labeled monoclonal antibodies has been an essential tool in preclinical biological research for decades. By binding selectively to target proteins or cellular structures, these labeled monoclonal antibodies enable high-sensitivity detection with minimal background noise. The properties that make optically labeled monoclonal antibodies ideal for imaging disease processes in preclinical mouse models or real-time imaging in the laboratory would significantly advance our understanding of drug-ligand interactions in human disease, as we and others have shown^5-7. We have adapted radiosynthesis modules to successfully manufacture optically labeled antibodies to advance our understanding of cellular functions, disease mechanisms and therapeutic responses in humans. The key difference relies on the scale of the process. In the radiosynthesis module, we use 1–3 mg of the antibody precursor, whereas in this Protocol, the labeling is performed at a larger scale, using 300 mg of monoclonal antibody. In addition, the layout and sequence used here are specifically tailored for optical labeling. This includes larger components, such as the reactor, syringes and purification cartridge, to accommodate the increased scale compared to those used in radiolabeling. We have previously shown the value of this strategy in understanding drug dosing, delivery and target engagement^8-17. To a lesser extent, optically labeled monoclonal antibodies are under investigation to improve surgical outcomes in oncology procedures¹⁴. These immunofluorescent probes, particularly those detected in the near-IR window (i.e., labeled with IRDye800CW or indocyanine green)^14-16, complement nuclear imaging modalities because they provide significantly higher-resolution imaging with limited background and real-time tumor visualization^11,18-23.Just as nuclear imaging using radiolabeled monoclonal antibodies can map disease processes, determine whole-body biodistribution and improve tumor detection, optically labeled therapeutic antibodies (OLTAs) can be used for single-cell resolution of monoclonal antibody–target engagement, biodistribution mapping of a drug within a tumor and intraoperative tumor detection²⁴ (Figs. 1 and 2).

Fig. 1 ∣. Optically labeled therapeutic antibody use for biodistribution analysis within the tumor.

Overview of the biodistribution study. Study patients received an infusion of optically labeled therapeutic antibody. One to two days after infusion, patients underwent surgical tumor resection. Fresh tissue samples were obtained from the primary tumor for homogenization to quantify the antibody–dye concentration in the tissue. Whole-tumor specimens were formalin-fixed and cut into 5-mm-thick tissue sections for macroscopic imaging. Subsequently, 5-μm histological slides were prepared from each tissue paraffin block made from the 5-mm tissue sections for microscopic imaging. Antibody uptake and distribution were measured from tissue homogenates and macroscopic and microscopic imaging. Reprinted from ref. 46, CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).

Fig. 2 ∣. Examples of OTLA usage for intraoperative tumor detection.

a–c, Patients were infused with anti-EGFR (cetuximab–IRDye800). In this phase II study, an OLTA was used to improve detection of surgical margins. Representative examples of a tumor-positive margin (a), a close margin (b) and a tumor-negative margin (c). In vivo fluorescence imaging shows sharply demarcated tumors compared to adjacent tissue (upper left images), and after excision, no fluorescence can be detected in the wound bed (upper right images). On the tissue slices, the tumor is delineated with a solid black line. Panel a shows a fluorescent spot with a signal-to-background ratio (SBR) of 4.0 on the excised specimen, corresponding to a tumor-positive margin (red arrowheads). Panel b shows a fluorescence spot with an SBR of 2.3, revealing a close margin of 2.2 mm (red arrowheads). The yellow arrow indicates a fluorescent lesion in the mucosa, which corresponds to the tumor spreading mucosally. In panel c, no fluorescent signal is seen in the margin, corresponding to a tumor-negative margin. Tumor tissue is demarcated with a solid black line on tissue slides. Reprinted from ref. 47, CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).

Development of the protocol

Antibody labeling with molecular tracers like fluorophores, radioisotopes, biotin or drugs is crucial to numerous fields of biological research, drug discovery and diagnostics. Direct methods of conjugating small molecules to antibodies are based on amine (peptide coupling)^25,26, thiol (maleimide alkylation)^25-27 or click (IEDA (inverse electron demand Diels–Alder)^28-30 or triazole²⁵) chemistry. These approaches can be performed by classical conjugation in solution or on-bead conjugation by capture and release flow-chemistry using magnetic protein beads³¹.

Currently antibodies are optically labeled in specialized clean room facilities to ensure sterility and comply with current good manufacturing practices (cGMP) standards for human use^8,10,15,32. However, the necessity of production in clean rooms and use of a manual process limits access to these imaging agents because of the high costs associated with constructing and maintaining such environments. Clean room maintenance is required to optimize cleanliness³³, differential pressure, temperature, humidity and airflow pattern³⁴ and contributes to high manufacturing costs. Moreover, this traditional manufacturing process requires large-batch (>10 g) synthesis when contract research organizations are engaged, which incurs additional time and labor, further escalating the development and production costs to >$1 million per batch.

Monoclonal antibodies conjugated to a cytotoxic drug or to an imaging agent have shown great potential in the clinical field of cancer treatment and diagnosis^35-37. Nevertheless, their implementation is limited by manual manipulations in the procedures for conjugation and manufacturing. To refine and simplify the complex and labor-intensive methodology, we have designed an automated optical labeling of monoclonal antibodies based on single-use components and using an automated synthesis module. Because the chemistry used for generating OLTAs and radiolabeling of monoclonal antibodies is similar (albeit at 10–100× scale), we leveraged a small-footprint radiosynthesis module, the iPhase MultiSyn, for the production of cGMP-compliant optically labeled antibodies.

The radiosynthesis module uses disposable cassettes and a simplified solid-phase purification cartridge. Single-use systems use disposable components such as a reactor, a single-use cassette, tubing and a purification cartridge. These components are pre-sterilized and are easily replaced between batches, eliminating the need for cleaning and validation between productions in accordance with cGMP standards. Furthermore, the manufacturing process for radiosynthesis modules is standardized for simple dissemination between centers.

Small-batch, automated production of OLTAs at a clinical dose scale (300 mg) reduced cost by 50% ($4,924 per dose to $2,500 per dose) and manufacturing time by 40%. Each batch is sufficient to treat four to five patients, based on a dosage of 50 mg per patient. Lowering the development and translation costs from >$1 million to <$100,000 enables academic research groups and startup companies to translate OLTAs for rapid evaluation without prohibitive resource allocation. This simplified, reduced-cost process may revolutionize the adoption of OLTAs for use in clinical research.

Building upon a recently reported automated method for the clinical production of a ⁸⁹Zr-labeled monoclonal antibody³⁸, we adapted this approach to develop an automated optical labeling method for the anti-epidermal growth factor receptor (EGFR) monoclonal antibody panitumumab based on our previous manual approach^8,39. The radiolabeling process incorporates automated fluid transfers, reaction buffer addition, size-exclusion-based purification and antibody formulation, which are similar steps to those used in our adapted method. However, the chemistry, MultiSyn module cassette, component size, reagents, reaction time and conditions required for radiolabeling a monoclonal antibody with ⁸⁹Zr differ significantly from those involved in optical labeling. We rationalized that the existing radiosynthesis module could be repurposed for our process with appropriate modifications and optimizations. This method is based on a single-use cassette that is coupled with a disposable size exclusion chromatography (SEC) cartridge, along with a 50-ml closed system centrifuge tube reactor (Fig. 3).

Fig. 3 ∣. MultiSyn cassette used for the automated optical labeling of panitumumab–IRDye800CW.

Designed single-use layout of the automated synthesis module for optical conjugation of panitumumab and IRDye800CW N-hydroxysuccinimide ester. The layout includes four manifolds, and each one has three positions, allowing the placement of 2 × 10-ml syringes for addition and transfer at positions 1 and 6, a reaction buffer vial at position 3, a column for purification at positions 4 and 11, a formulation buffer at position 5, two more syringes for reagent (panitumumab and IRDye) addition at positions 8 and 10, a reaction vessel at position 9 and a final product vial at position 12. mAb, monoclonal antibody.

We demonstrate this process by manufacturing panitumumab–IRDye800CW under cGMP conditions by using an iPhase MultiSyn module. Panitumumab was purchased from the pharmacy and conjugated via a classical peptide coupling method, using the amino groups on the lysine residues and the N-hydroxysuccinimide (NHS) ester group of the IRDye800 (Fig. 4). The reaction conditions for conjugation were optimized, resulting in a final product with an optimal degree of labeling (DOL) as well as high yield and purity (Fig. 5d and Supplementary Fig. 3).

Fig. 4 ∣. Schematic conjugation reaction of the panitumumab–IRDye800CW.

Amide bond formation conjugation of the primary amine groups of the panitumumab antibody with the NHS ester group of the IRDye800CW dye.

Fig. 5 ∣. SEC-HPLC chromatograms at 280 and 780 nm.

a–d, Chromatograms of panitumumab (a), IRDye800CW NHS ester (b), IRDye800CW carboxylate (c) and panitumumab–IRDye800 (d). mAU, milli-absorbance units; Ref, reference; Sig, signal.

After demonstrating the feasibility of producing optically labeled anti-EGFR monoclonal antibodies, we confirmed broad application of the technique by using an anti-PD-1antibody (nivolumab). Nivolumab–IRDye800CW was produced with the same yield, labeling ratio and purity, suggesting that our proposed automated synthesis has broad application for labeling therapeutic monoclonal antibodies with optical dyes.

Comparison with other methods

This Protocol, originally developed for panitumumab, has been designed as a generalizable approach to overcome the difficulties of using manual methods to optically label therapeutic monoclonal antibodies. To this end, Table 1 reviews the major pitfalls of the current manual manufacturing and details the corresponding improvements offered by automated bioconjugation synthesis.

Table 1 ∣.

Side-by-side comparison of the manual and automated optical labeling process of monoclonal antibodies

Item	Manual	Automated	Specifications
Process	Manual operator movements	MultiSyn radiosynthesizer	Automated processes minimize the risk of human error, ensuring that the optical labeling of monoclonal antibody is performed in a consistent and reproducible manner with reduced time and high accuracy. In addition, and of great importance, the automated system is an enclosed cassette that maintains a low bioburden, eliminating the need for the manufacturing operations in a clean room
Purification	Reusable column	Single-use cartridge	Disposable SEC cartridge is used to eliminate the need for cleaning and sterilizing a reused purification column, as well as the validation of cleaning procedures (including chemical purity, bioburden, sterility, etc.)
QC	Gas chromatography	Not required	Unlike the manual process, the automated method does not involve re-used SEC columns that require cleaning with organic solvents and subsequent characterization by gas chromatography. Instead, single-use cartridges are used
Time	7 h	4 h	The automated synthesis is exempt from SEC column conditioning, manual purification or gas chromatography analysis. MultiSyn allows for column conditioning to occur simultaneously with the conjugation reaction, ensuring optimized elution and consistent collection of the final product. This approach eliminates the need for additional characterization of product purification and column cleaning
Total required infrastructure footprint for manufacturing	400–800 square feet	12 square feet	Manual synthesis requires a clean room of 400–800 square feet, whereas the automated module needs only 12 square feet of space
Annual maintenance	$150,000	$8,000	Maintaining and monitoring an ISO 5 clean room for manual drug production to ensure compliance with cGMP regulation can cost significantly more than maintaining a MultiSyn module used for automated production

Applications of the method

This automated approach using the MultiSyn module, originally developed for labeling of panitumumab, is applicable for the optical labeling of different monoclonal antibodies. To demonstrate broader applicability, the process was successfully applied to produce a small-scale batch of nivolumab–IRDye800CW. Using the same conditions of panitumumab–IRDye800CW production as reported herein, the results (Table 2) demonstrate that the conjugation of nivolumab with the IRDye800CW NHS ester resulted in the fluorescently labeled anti-PD-1 product, with excellent yield, optimal dye-to-protein (D/P) ratio and high purity (Supplementary Figs. 6-8).

Table 2 ∣.

Results of the automated optical labeling of nivolumab–IRDye800CW

Initial mass (mg)	Mass collected (mg)	Concentration (mg ml−1)	Yield (%)	Purity (%) at 280 nm	Purity (%) at 780 nm	IRDye800 (%) at 780 nm	D/P ratio	pH	HMWS (%) at 280 nm	LMWS (%) at 280 nm
40	38.74	1.49	96	97.3	99.8	0	1.69	7.4	0.3	2.4
160	154.57	5.16	96	98.8	98.7	1.1	1.97	7.4	0.2	1.7

HMWS, high molecular weight species; LMWS, low molecular weight species.

In addition to the production of panitumumab–IRDye800CW and nivolumab–IRDye800CW, this automated process can be applied to optical labeling of any FDA-approved monoclonal antibodies formulated without amino acid (e.g., L-histidine and L-arginine), such as cetuximab, bevacizumab, rituximab and ipilimumab. Moreover, this approach may provide an alternative method for pilot-scale manufacturing of antibody-drug conjugates currently in clinical development, including cetuximab sarotalocan, which has been approved by the Pharmaceuticals and Medical Devices Agency (PMDA; Japan)⁴⁰. When applying this technique to label other antibodies formulated at concentrations different from panitumumab, optimization may be necessary to adjust parameters such as pH, loading volume and purification conditions to ensure the efficient production and collection of the final conjugated product.

Optimizations of the optical labeling

Currently, fluorescently labeled anti-EGFR monoclonal antibodies, such as panitumumab⁸ and cetuximab¹⁰, are manually produced by using a 2.3 equivalent molar ratio of IRDye800CW NHS ester, usually incubating at room temperature for 2 h with either a carbonate or phosphate buffer and at a pH of 8.5. With these conditions as a starting point, we performed an automated production as presented in Table 3, Entry 1. We dissolved 2.88 molar equivalents of the dye in the reaction buffer (1.0 M K₂HPO₄) at a concentration of 15 mg ml⁻¹ (5 mg in 0.333 ml of reaction buffer). To this solution, panitumumab was added, followed by the reaction buffer (0.1 ml of 1.0 M K₂HPO₄ 1 ml of panitumumab⁻¹, 20 mg ml⁻¹). After 2 h of mixing via periodic N₂ bubbling, the reaction mixture was loaded and passed through a P-6 SEC column. The panitumumab–IRDye800CW was isolated with a 0.79 D/P ratio, as shown in Fig. 5d.

Table 3 ∣.

Optimization of the optical labeling conjugation reaction of the panitumumab with IRDye800CW NHS ester

Entry	Labeling type	Scale (mg)	Order of addition		IRDye800 NHS ester				Reaction mixture pH	D/P ratio
			Reagent 1	Reagent 2	Reagent 3	IRDye solvent	IRDye Eq	IRDye (mg ml−1)		15 min	60 min	120 min
1a	Automated	200 ± 100	IRDye	1 M K2HPO4	Pan	1 M K2HPO4	2.88	15	ND	ND	ND	0.76 ± 0.04
2	Automated	100	IRDye	1 M K2HPO4	Pan	1 M K2HPO4	2.88	15	8.8	0.8	0.8	0.8
3	Manual	50	IRDye	1 M K2HPO4	Pan	H2O	2.88	15	8.12	1.28	1.28	1.28
4	Manual	50	Pan	1 M Na2HPO4	IRDye	H2O	2.88	15	8.13	1.23	1.23	1.23
5	Manual	50	Pan	1 M Na2HPO4	IRDye	H2O	2.88	15	8.01	1.34	1.34	1.34
6	Manual	50	Pan	1 M Na2HPO4	IRDye	H2O	2.88	15	8.04	1.66	1.66	1.66
7	Manual	50	Pan	1 M Na2HPO4	IRDye	H2O	5.6	15	7.9	2.87	2.91	2.93
8	Manual	50	Pan	1 M Na2HPO4	IRDye	H2O	5.6	5	7.95	2.7	2.7	2.71
9	Automated	50	IRDye	1 M K2HPO4	Pan	H2O	5.6	5	8.83	ND	2.62	ND
10	Automated	300	IRDye	1 M K2HPO4	Pan	H2O	5.6	15	ND	ND	2.6 ± 0.3	N/A
11a	Automated	300	IRDye	1 M K2HPO4	Pan	H2O	4	10	ND	ND	1.8 ± 0.15	N/A

N/A, not applicable; ND, not determined; Pan, panitumumab. For all the automated runs, the final product was isolated with a yield of 96% ± 1%.

^aNumber of tests (n = 3).

The optimal D/P ratio needed for clinical use of panitumumab–IRDye800CW to ensure an appropriate half-life, comparable binding affinity to the unlabeled panitumumab and a high image signal with a good tumor-to-background ratio is ~2.0 ± 1.0 (ref. 41). Entry 1 does not meet the required acceptance criteria. To improve the product output, multiple optimizations were performed to achieve an optimal D/P ratio. We hypothesized that the automated conjugation reaction might require a longer time than the manual one, because of inefficient mixing. We monitored the conjugation reaction by HPLC to determine the optimal reaction time. The results are shown in Table 3, Entry 2 and in Supplementary Fig. 1; the D/P ratio is 0.8 for 15 min and remains constant for 2 h. Thus, the conjugation reaction is complete in 15 min, which is shorter than has previously been reported^8,10.

To further optimize the D/P ratio, the conjugation was performed manually without purification, and the reaction was evaluated by HPLC. The low DOL obtained in Entries 1 and 2 in Table 3 is associated with the hydrolysis of the NHS ester and the decomposition of the IRDye800. As observed in the chromatogram in Fig. 5c, the IRDye in PBS presents as a single peak at 26.7 min; however, once dissolved in 1.0 M K₂HPO₄ with a pH of 9.12, multiple peaks were detected between 24 and 28 min in the chromatograms (Supplementary Fig. 1). We believe that the NHS ester has been decomposed before reacting with the antibody. This result is consistent with the literature⁴², which shows that after 5 min of incubation, 20% of the NHS ester has already been hydrolyzed in PBS with a pH of 8.5. For this reason, the IRDye solvent was changed to water, which produced an improved DOL of 1.28 (Entry 3). The order of reagent addition has no effect on the DOL (Entry 4). Our results demonstrate that using water does not decompose the IRDye, which was observed with a single peak at a retention time of 26.7 min at the 780-nm channel (Supplementary Fig. 2). The pH of the reaction mixture was lowered to 8.01 by using 1.0 M Na₂HPO₄ with a pH of 8.83, resulting in a minor improvement of DOL (Entry 5). The IRDye800 NHS ester, a lyophilized powder, was weighed out from a vial of 5 mg and then dissolved for Entries 2–5. In Entry 6, it was directly dissolved and then partitioned, which produced a better DOL of 1.66. Bevacizumab–800CW is a fluorescently labeled anti-VEGF-A monoclonal antibody that has a similar molecular weight to panitumumab. Despite using 4 equivalents of the IRDye800 NHS ester, bevacizumab–800CW was produced with a 1.59 (±0.04) D/P ratio³². Using this as guidance, the IRDye800’s equivalents were doubled to 5.6, producing panitumumab–IRDye800CW with a D/P ratio of 2.9. Furthermore, the purification of the final drug product from the reaction mixture remained unaffected by the increase in the dye amount (Entry 7). We assessed the effect of the concentration of the dye on the DOL. The dye was diluted in water from 15 to 5 mg ml⁻¹, and the resulting D/P ratio was similar (±0.2) (Entry 8).

We translated these optimal conditions to the automated production. Unexpectedly, the 1 M Na₂HPO₄ used in the current manual process was found to be unsuitable for automation because of precipitation at room temperature (20–23 °C), causing clogs in the lines and leading to the cassette tubing bursting under flow pressure. In Entry 9, we automatically reproduced the conditions from Entry 8 by using 1 M K₂HPO₄ at a scale of 50 mg of panitumumab, and the product was isolated with a similar result. Finally, a larger scale of 300 mg was performed, and the final drug product panitumumab–IRDye800CW was produced with an optimal D/P ratio and an excellent yield. It is important to note that the yield of the conjugation process has been optimized in Entry 1 and enhanced from 61% to 96%. During the loading and purification step detailed in the automated labeling part of the procedure, the reaction mixture is initially loaded onto the SEC cartridge, with the load directed to waste. Collection begins only after the entire compound has been loaded onto the cartridge, which results in a 61% yield. However, we observed that the desired green optically labeled product band starts to elute from the column during the final 5 ml of the load phase. As a result, the automated sequence has been adjusted to initiate collection during the loading step to enhance the yield of the desired product up to 96%. As a final optimization, the dye equivalency was reduced to 4 (2 × 5-mg vials at the 300-mg scale) to give a D/P ratio of 1.8 ± 0.15 (n = 3; Entry 11).

Estimating the degree of labeling

The DOL, also known as ‘D/P ratio’, represents the average number of dye molecules that are conjugated to the antibody. Prior optimization studies revealed issues concerning both under-labeling and over-labeling of antibodies⁴³. Under-labeling does not provide sufficient image signal for effective tumor visualization and diagnosis. On the other hand, over-labeling with fluorophores can alter antigen binding characteristics, reduce circulation half-life, decrease fluorescence via quenching mechanisms and lower the hydrophilic property of the monoclonal antibody, which may produce dimers and aggregates. To overcome the issues associated with under- and over-labeling, the optimal D/P ratio of anti-EGFR antibody falls within the range of 1–3 dyes per antibody molecule^8,10,14,32. This intrinsic property of the purified panitumumab–IRDye800CW conjugate, as shown in Fig. 5d, was determined by using standard calibration curves (Supplementary Fig. 4a,b) of both panitumumab (Fig. 5a) and IRDye800 carboxylate (Fig. 5b), which were obtained by HPLC. The equations used for these calculations are detailed in Supplementary Fig. 5 and described comprehensively in the Supporting Information.

Determination of the DOL of panitumumab–IRDye800

1. In the ISO 5 environment, using aseptic technique, withdraw a 0.2-ml aliquot from the bulk solution of the purified conjugate pantimumab–IRDye800.

2. Dilute 100 μl of the produced pantimumab–IRDye800 in 900 μl of 1× PBS and transfer it to an autosampler vial.

3. Inject 50 μl into the SEC–high-pressure liquid chromatograph.

4. The peak area at 14.3 min of the signals at 280 and 780 nm were measured. The mass of the panitumumab was obtained by solving Equation 1 in Supplementary Fig. 5, and the IRDye800 was directly extrapolated from its calibration curve by using Equation 2 in Supplementary Fig. 5.

5. It is important to note that the percent contribution of IRDye800CW carboxylate at 280 nm is 0.03. This value was determined by dividing the average peak area of the dye at 26.6 min at 280 nm by its average peak area at 26.6 min at 780 nm.

6. The resulting mass was divided by the molecular weight of both reagents (M1 = 145,000 g mol⁻¹ and M2 = 1,091.1 g mol⁻¹) to convert them into moles. These values were then used to estimate the DOL by solving Equation 3 in Supplementary Fig. 5 and to finalize the Supplementary Table 1.

Quality control

The quality control (QC) procedure of the fluorescently labeled monoclonal antibody is estimated to take up to 6 work hours. The drug produced via automated manufacturing meets the release criteria previously reported^14,32 and listed in Table 4.

Table 4 ∣.

List of QC tests of the optically labeled monoclonal antibody

QC test	Description	Acceptance
Appearance	Visual inspection for color and particulates	Green-blue tinted, clear to slightly opalescent liquid, free of visible particulates
Sub-visible particulates	USP <787>/<788>	PQIT
Chemical identity	HPLC consistent with guidelines of USP<621> chromatography subsection high-pressure liquid chromatography	Retention matches reference standard with a relative retention time of 0.9–1.1
	SDS-PAGE conducted under both reducing and non-reducing conditions to estimate the molecular weight and determine the densitometry of bands	Molecular weight and densitometry of bands that constitute >5% relative to the unlabeled standard
Concentration	HPLC consistent with guidelines of USP<621> chromatography subsection high-pressure liquid chromatography	5 ± 0.5 mg ml−1
D/P ratio	HPLC consistent with guidelines of USP<621> chromatography subsection high-pressure liquid chromatography	1–3
Potency	HPLC consistent with guidelines of USP<621> chromatography subsection high-pressure ciquid Chromatography	≥50% total binding fraction
Chemical purity	HPLC consistent with guidelines of USP<621> chromatography subsection high-pressure liquid chromatography	≥90% monomer
Free dye	HPLC consistent with guidelines of USP<621> chromatography subsection high-pressure liquid chromatography	≤10% free dye at 780 nm
Residual NHS	HPLC consistent with guidelines of USP<621> chromatography subsection high-pressure liquid chromatography	Report
pH	pH	7.4 ± 0.5
Bacterial endotoxin levels	LAL USP <85>	≤5 EU ml−1
Sterility	USP sterility test (USP <71>)	No growth observed in 14 d

EU, endotoxin units; LAL, Limulus amebocyte lysate; PQIT, periodic quality indicator test; USP, United States Pharmacopeia.

Limitations

• The current protocol is restricted to the use of only those monoclonal antibodies that do not require pretreatment processes such as desalting or pre-purification (e.g., monoclonal antibodies formulated with histidine).

• The final drug product was successfully produced with an excellent yield (96% ± 1%) for scales of 300 mg of panitumumab. For these scales, we used a 50-ml Bio-Gel P-6 (SEC) desalting cartridge. Scaling up beyond 300 mg would exceed the loading volume of the P-6 cartridge and would reduce the purity of the final product. This might be solved by incorporating multiple 50-ml desalting cartridges in series, although it has not been tested.

Expertise needed to implement the protocol

A significant benefit of this approach is that most steps of the protocol are well known to individuals familiar with radiosynthesis modules, cGMPs and aseptic manufacturing. The radiosynthesis module requires only installing the cassette, handling chemicals and validating the automated sequence. Conducting QC of the final drug product, including HPLC, filtration, formulation and sterility tests, requires specialized training in a radiochemistry core or similar pharmaceutical facility. Determining the final compound identity involves basic biochemistry techniques such as gel electrophoresis.

Materials

Reagents

▲ CAUTION It is important to follow standard laboratory safety precautions, including wearing personal protective equipment when handling these materials.

Analytical methods

• Milli-Q water (1 liter)

• Sodium azide, 0.65 g (Sigma-Aldrich, cat. no. 26628-22-8)

• 10× PBS, 5 ml (Cytiva, cat. no. SH30258.01)

• 5 M sodium chloride solution, 30 ml (Corning, cat. no. 46-032-CV)

• 2× NuPAGE 4–12%, Bis-Tris, 1.0-mm mini protein gel, 10 wells (Thermo Fisher, cat. no. NP0321BOX)

• 120-μl Invitrogen Mark12 unstained standard (Thermo Fisher, cat. no., LC5677)

• 120-μl Invitrogen NuPAGE lithium dodecyl sulfate sample buffer (4×) (Thermo Fisher, cat. no. NP0007)

• 500-ml Invitrogen NuPAGE MOPS sodium dodecyl sulfate running buffer (20×) (Thermo Fisher, cat. no. NP0001)

• 9-μl Invitrogen reducing agent NuPAGE 10× (Thermo Fisher, cat. no. NP004)

• 150-ml Invitrogen SimplyBlue SafeStain (Thermo Fisher, cat. no. LC6060)

• Saline, 0.9% (wt/vol) (Cytiva, cat. no. Z1377)

Manufacturing

• Panitumumab, 300 mg (Amgen, NDC 55513-954-01)

• 1.0 M potassium phosphate dibasic aqueous solution, 6 ml (Millipore-Sigma, cat. no. P8584)

• 50 mM sodium phosphate with 2% mannitol (wt/vol), pH 7.4, 2 × 250-ml bottles (Teknova, cat. no. 22505)

• IRDye800CW NHS ester, 10 mg (LI-Cor, part no. 929-70020)

• IRDye800CW carboxylate, 5 mg (LI-Core, part no. 929-09406)

• Cell culture-grade water, 1 ml (Corning, cat. no. 25-055-CI)

• Bio-Scale Mini with Bio-Gel P-6 (SEC) desalting cartridge, 50 ml (Bio-Rad, cat. no. 7325312)

  ▲ CRITICAL This protocol was optimized for the specific dimensions of this cartridge; substitution is not recommended.

Equipment

• Sterile, 50-ml, closed-system centrifuge tube, self-standing with dip tube

• (Corning, cat. no. 11705)

• Luer lock sterile two-part NORM-JECT syringes, 3 × 10 ml (Henke Sass Wolf, cat. no. 8300027527)

• Luer lock sterile three-part syringe, 1 × 3 mL, 20 gauge, 1 inch (BD, cat. no. 309579)

• Luer lock sterile three-part syringes, 20 ml (BD, cat. no. 302030)

• 18-gauge × 6-inch spinal needle (Medline, cat. no. PAIN8033)

• 4 × 21-gauge 2-inch sterile needles (BD, cat. no. 305196)

• 2 × green needles (BD, cat. no. 305129)

• 1 × 18-gauge 1 1/2-inch sterile needle (BD, cat. no. 305196)

• 8 × sterile, filtered vent needles (International Medical Industries, cat. no. 31-20)

• 8 × Pall filters, 0.22 μm (Pall, cat. no. AEF1NTE)

• 100-ml sealed sterile vial (ALK, cat. no. SEV100)

• 10-ml sealed sterile vial (ALK, cat. no. SEV1020)

• 2 × sterile septum caps (ThermoFisher Scientific, cat. no. 02-923-20)

• MultiSyn cassette, optical monoclonal antibody radiolabeling (IPHASE technologies, cat. no. MSH-310)

  ▲ CRITICAL This cassette assembly contains all of the materials in a pre-assembled low-bioburden set.

Manufacturing

• IPHASE technologies MultiSyn radiosynthesizer

  ▲ CRITICAL The procedure was optimized on this module. Substitution would require re-optimization.

• External heating block (Fisher Scientific, cat. no. 14-955-241)

Equipment setup

Analytical methods

• HPLC system equipped with a 280- and 780-nm channel detector

• HPLC column: Superdex 200 Increase 10/300GL, column length × i.d.: 30 cm × 10 mm, 8.6-μm particle analytical column (Cytiva, cat. no. 28-9909-44)

  ▲ CRITICAL Choose the appropriate SEC column as a function of the molecular weight of the protein being labeled. This column is well suited for IgGs.

• Glass vacuum filtration distillation apparatus (2,000 ml), including a 1-liter graduated funnel filter flask, a 10-μm pore-size clamp compatible with 42–60-mm membrane filter paper and 0.45-μm membrane filter paper.

• NanoDrop One UV-visible system (Thermo Scientific, cat. no. AZY1704793)

• UV-visible spectrophotometer to scan in the range of 200–800 nm (Beckman Coulter, cat. no. DU-730)

• Invitrogen PowerEase Touch 120 W power supply and mini gel tank (Thermo Fisher, cat. no. PSC120MB)

• Eppendorf Thermomixer C (Marshal Scientific, cat. no. EP-5382000023)

• LSE mini microcentrifuge (120 V; Corning, cat. no. 6770)

• 1–200-μl, round, 0.5-mm-thick gel-loading pipette tips (Aldrich, cat. no. CLS4853)

• Two Owl gel-staining boxes (Thermo Fisher, cat. no. OW-GSB-3)

• Invitrogen gel knife (Thermo Fisher, cat. no. EI9010)

• Two Invitrogen blotting tweezers (Thermo Fisher, cat. no. B1002)

• Fisher Vortex Genie 2 (Thermo Fisher, cat. no. 12-812)

• LI-COR Odyssey CLx Imager (LI-COR, CLx Imager)

• 2-ml microcentrifuge tubes (Fisher Scientific, cat. no. 02-681-321)

• Compressed air supply and calibrated pressure gauge (used for the filter-integrity test)

MultiSyn module

• Turn on air and nitrogen to the module, check that the nitrogen pressure is >500 p.s.i. and ensure that the regulator is set to 15.0–20.0 p.s.i.

• Remove any materials from previous use on the MultiSyn module. Dispose of spent syringes and buffer vials or flasks along with the reactor and purification column.

• Empty the waste bottle.

• Turn on the isotherm dry bath and set the temperature at 25 °C.

Procedure

Cassette assembly

● TIMING 5 min

• 1Thoroughly and visually inspect the manifold of the MultiSyn module (Fig. 6) for visible hairline cracks on the stopcock manifolds before each synthesis run.
• 2Ensure that the rotating taps are facing away and not to the front. Verify that all taps are straight and rotated down; otherwise, the cassette cannot be installed.
• 3Remove the 18.5-cm tube from position 9 and connect it to the 50-ml reactor female port. (First remove the filter from the reactor.)
• 4Connect the long, thick tube from the reactor to position 9.
• 5Place the 10-ml two-piece syringes on the cassette at positions 1 and 6, ensuring a good connection.

Fig. 6 ∣. Fully assembled MultiSyn cassette and reactor used for panitumumab–IRDye800CW production.

This image represents the mounted cassette installed into the MultiSyn module, as illustrated in the designed layout in Fig. 4.

Initiating the MultiSyn software

● TIMING 4 min

• 6.Log on to the MultiSyn software on the operating laptop with your credentials.
• 7.From the operating laptop, select ‘Download Recipe’ from the main menu.
• 8.Choose the sequence ‘Panitumumab-IRDye800 v1.0’.
• 9.Once the software is downloaded and checked, click ‘Ok’.
• 10.Click ‘Start’ and enter the batch number as ‘YYMMDD_PAN800’.
• 11.Following the instructions in the recipe, remove any remaining cassettes or other materials from previous syntheses.

Installing the cassette onto the module

● TIMING 5 min

• 12.Start installing the top manifolds in the correct positions (Fig. 6) and secure the syringes at positions 1 and 6 to the plungers. Make sure that all the tubing is facing forward, to avoid punctures.
• 13.Install the bottom manifolds and make sure that all tubing is facing forward.
• 14.Connect the thin tube from the reactor to the central reactor gas/vacuum connection.
• 15.Connect the waste and gas connections from manifolds 1, 2, 3 and 4 to their corresponding ports on the module, as shown in Figs. 4 and 6.
• 16.Close all magnetic clamp arms to secure the cassette to the module.
• 17.Place the reactor in the isotherm dry bath at 25 °C.

Sequence check test

● TIMING 5 min

• 18.Click ‘Next’ in the software when done mounting.
• 19.After that, the system will check the connection for leaks. If any of the steps in the check test fail, use the error message displayed by the software to identify the cause of the failure and resolve the leak. Once the leak is resolved, repeat Steps 18 and 19 to ensure that the system is functioning properly before continuing to Step 20.

◆ TROUBLESHOOTING

Placing reagents, buffers and vials onto the cassette

● TIMING 25 min

• 20.Connect a sterile 100-ml bulk vial with vent filter and a Pall sterilizing filter to the tubing at position 12 on the MultiSyn cassette.
• 21.
  For the urification column, complete the following steps:

  • Connect the tubing at position 4 to the female (top) side of the P-6 column
  • Connect the tubing at position 11 to the male (bottom) side of the P-6 column
  • Place the P-6 column into the holder, ensuring that the tubing is free of kinks or pinches
• 22.
  For the reaction buffer, complete the following steps:

  • Install a filtered vent needle to the 10-ml sterile empty vial
  • Using a 10-ml syringe with needle, measure out 6 ml of 1 M potassium phosphate buffer and add it to the 10-ml vial
  • Remove the vented filter needle
  • Install the buffer vial at position 3 on the MultiSyn cassette
• 23.
  For the formulation buffer, complete the following steps:

  • Obtain the 250-ml bottle of the formulation (50 mM sodium phosphate with 2% mannitol (wt/vol), pH 7.4)
  • Remove the cap from the bottle and replace it with the septum cap
  • Install a filtered vent needle through the septum cap
  • Install the 16-gauge × 6-inch spinal needle all the way to the bottom of the bottle
  • Connect the spinal needle to the tubing from position 5 on the MultiSyn cassette
• 24.
  For the panitumumab syringe, complete the following steps:

  • Install filtered vent needles to the three panitumumab vials
  • Attach a green needle to the syringe
  • Using the 20-ml three-part syringe, pull up 15 ml of the panitumumab solution from the three vials
  • Pull an additional 5 ml of air from the final panitumumab vial into the syringe (15 ml of solution and 5 ml of air in the syringe)
  • Remove the needle from the syringe
  • Attach the syringe at position 10 on the MultiSyn cassette
• 25.
  For the dye syringe, complete the following steps:

  • Just before starting the sequence, using a calibrated pipettor, add 0.5 ml of cell culture water to both IRDye800CW-NHS ester vials
  • Recap the vials. Swirl or vortex for 10 s to ensure dissolution of the dye
  • Equip the 3-ml syringe with a green needle
  • Using the 3-ml syringe, pull up the dye solution from each vial
  • Pull the syringe up to 3 ml with air (for a total of 1 ml of dye solution and 2 ml of air)
  • Attach the syringe to position 8 on the MultiSyn cassette
• 26.Verify that you have mounted the cassette and that the reagents and vials are as presented in Fig. 6.
• 27.Click ‘Next’ in the software; the system will start the automated synthesis.

Automated labeling

● TIMING 1 h 40 min

• 28.From this point, the sequence was created to provide an automated addition of the reagents, followed by equilibration of the SEC, reaction mixture homogenizing, purification and collection of the final product. Steps 29–35 are executed automatically and require no user input.
• 29.The reactor is placed under vacuum. The IRDye800CW NHS ester solution is drawn and transferred from the syringe at position 8, following the reactor’s vacuuming. After that, the reactor is depressurized with a low flow of nitrogen.
• 30.Once the reaction buffer vial at position 3 is pressurized with a low-flow nitrogen, 2.0 ml of the reaction buffer is drawn up by using a syringe at position 1 to prompt the line and send to the waste bottle.
• 31.Using the same syringe at position 3, another 1.5 ml of the reaction buffer is pulled up and then added to the reactor to adjust the pH mixture and achieve the optimal pH of 8.3 ± 0.5 for the conjugation.
• 32.The reactor is placed again under vacuum, and 15 ml of panitumumab (300 mg) is added to the reactor.
• 33.To enable a complete transfer of the reagents to the reactor, the transfer lines are automatically flushed with nitrogen after each addition step.
• 34.During the conjugation reaction, using the syringe at position 6, the SEC cartridge is conditioned with 10×10 ml of the formulation buffer.
• 35.The lines between position 1 and position 6 and between position 1 and position 7 are dried by using a dual nitrogen/vacuum flow after the SEC cartridge has been equilibrated. This prevents the formulation buffer, which contains sugars, from being transferred to the reactor while the reaction mixture is bubbling. Consequently, absence of formulation buffer in the reactor prevents undesired susceptible trans-esterification with the IRDye800 NHS ester, which may cause a low DOL.
• 36.The conjugation reaction is allowed to occur at 25 °C for 1 h. To ensure that the solution is homogenized, nitrogen is bubbled through the reaction mixture for 30 s after every 10 min of reaction.
• 37.Once the conjugation reaction synthesis is completed, the crude product must be purified by using SEC to eliminate the excess free dye.
• 38.The crude Pan800 product is loaded on the SEC column by using the syringe at position 1. The 17.5 ml of the green mixture is passed through the SEC column, and the flow-through is sent to the waste bottle. When only 2 ml remain in the syringe at position 1, the stopcock valve at position 11 is switched from the waste position to the final product vial. The process continues by rinsing the syringe at position 1 and the lines with 4 ml of formulation buffer. This rinse is then loaded onto the SEC column and collected in the final product vial at position 12.
• 39.The trapped Pan800 conjugate is eluted with 2 × 10 ml of the formulation buffer by using the syringe at position 6. The pure Pan800 is collected in a total volume of 26 ml in the final product vial at position 12, followed by flushing the final product tubing with a low flow of nitrogen for 20 s.
• 40.Finally, remove the filtered vent needle from the final product vial. Disconnect the vial from the MultiSyn module. If skipping Step 41, proceed without storing the vial. Otherwise, store it overnight at 2–8 °C in the dark for dilution and/or use.
• 41.(Optional) At this stage, the user has the option to perform dilution, prepare aliquots or perform other additional processing, if desired, in an ISO 5 aseptic environment. This step is optional to allow flexibility in the workflow, because QC tests may either be conducted immediately after manufacturing or be postponed to the following day.

  ■ PAUSE POINT The procedure may be paused here for up to 24 h. If pausing, store the bulk vial at 2–8 °C protected from light.

End of automated labeling

● TIMING 5 min

• 42.Upon completion of the automated synthesis, click ‘Abort’ followed by ‘OK’ to shut down the MultiSyn software.
• 43.Close the nitrogen supply and turn off the isotherm dry bath.
• 44.Finally, if Step 41 was not performed, using aseptic techniques, transfer the bulk vial to the laminar flow hood ISO 5 area for QC. If any product dispensing or preparation of aliquots for human use is desired, it must be performed per local regulations (i.e., FDA, Board of Pharmacy or other national regulatory authorities).
• 45.Click ‘Abort’ after the synthesis is complete.
• 46.Turn off the nitrogen and the isotherm bath.

Troubleshooting

Recommendations for troubleshooting potential problems that may occur when running the automated bioconjugation reaction are provided in Table 5.

Table 5 ∣.

Troubleshooting table

Step	Problem	Possible reason	Solution
19	The sequence check test failed	Leak in the cassette	The check sequence test was conducted so that the system examines each connection separately and individually. Thus, if any step in the test fails, use the error message displayed by the software to identify the cause of the failure
			Ensure that the nitrogen gas regulator is set to 15.0–20.0 p.s.i
			Inspect the stopcock manifolds for hairline cracks before installing the cassette
			Verify that all tubing and the reactor are connected to the module and that the manifolds are securely tightened
			Use a new cassette
		Clog in the system	Identify the clog
			If it is in the gas line, run a high nitrogen flow for a few minutes to clear the blockage
			If it is in the vacuum line, connect 42-cm tubing to a 20-ml syringe and purge the clogged vacuum system with 3 × 20 ml of water by turning the vacuum on in the MultiSyn software and directing the purge to the waste bottle. If the clog persists, connect the 42-cm tubing between the clogged vacuum line and the gas outlet and run a high nitrogen flow for a few minutes to clear it
			Use a new cassette
QC	Low D/P ratio	IRDye800 NHS ester was hydrolyzed or decomposed	Use reagent immediately after dissolution in water
			The reagent was dissolved in the reaction buffer K2HPO4 instead of water, which resulted in its decomposition at basic pH

Timing

Panitumumab–IRDye800CW automated manufacturing is estimated to take 4 h, followed by 6 h of QC.

Steps 1–5, cassette assembly: 5 min

Steps 6–11, logging in to the MultiSyn software and loading the sequence: 4 min

Steps 12–17, mounting the cassette into the module: 5 min

Steps 18–19, sequence check test: 5 min

Steps 20–27, reagent preparation and installment onto the module: 25 min

Steps 28–46, automated labeling: 1 h 40 min

Anticipated results

In this Protocol, we describe an automated approach for optical labeling of monoclonal antibodies using an IPHASE MultiSyn module. We have conducted a validation process to ensure the quality of the product for research clinical applications and to confirm the reproducibility of the automated synthetic procedure. Table 6 presents the result of process validation (PV) of three consecutive batches of panitumumab–IRDye800CW production.

Table 6 ∣.

QC results of the PV of three consecutive batches of panitumumab–IRDye800CW production

QC test	Specification	Quality run 1	Quality run 2	Quality run 3
Appearance	Blue-tinted, clear to slightly opalescent liquid free of visible particulates	Pass	Pass	Pass
Sub-visible particulates	≤6,000 particles ≥10 μm 10-ml bag−1, ≤600 particles ≥25 μm 10-ml bag−1	86 (≥10 μm); 34 (≥25 μm)	99 (≥10 μm); 18 (≥25 μm)	53 (≥10 μm); 23 (≥25 μm)
Identity (HPLC)	RRT: 0.9–1.1	1.0	1.0	1.0
Identity (SDS-PAGE)	Conforms to standard	Conforms	Conforms	Conforms
Strength/potency	≥50% EGFR binding fraction	>99%	>99%	>99%
Concentration	Absorbance at 280 nm; 5 ± 0.5 mg ml−1	4.9 mg ml−1	5.1 mg ml−1	5.1 mg ml−1
D/P ratio	1–3	1.75	1.74	2.00
Purity	Monomer purity at 280 nm ≥90%	96%	97%	97%
Residual IRDye800	Free dye ≤10% at 780 nm	1.23%	0.93%	2.81%
Residual NHS	Report	0.125 μg ml−1	0.281 μg ml−1	0.150 μg ml−1
pH	7.4 ± 0.5	7.4	7.5	7.4
Filter integritya	Meets manufacturer specifications	Pass	Pass	Pass
Sterility	USP <71>	No growth	No growth	No growth
Bacterial endotoxin levels	≤5 EU ml−1	<1.00 EU ml−1	<1.00 EU ml−1	<1.00 EU ml−1

RRT, relative retention time.

^aThis applies to sterilizing filters used for each dose aliquot.

The three qualification runs yield the final product, panitumumab–IRDye800CW, which appears as a green-blue-tinted liquid, free of visible particulates and meeting all investigational new drug release criteria.

Analytical HPLC is the gold standard technique used to evaluate the final product’s quality, including its identity, EGFR-binding potency, concentration, labeling efficiency, chemical purity and residual impurities in the final dose. The product was identified by comparing the HPLC retention time (UV monitoring at 280 nm) of the conjugate (Fig. 5d) with that of non-labeled panitumumab (Fig. 5a). Identity is also assessed by SDS-PAGE, in which the estimated molecular weight and the densitometry of the proteins conform to the non-labeled panitumumab standard (Supplementary Fig. 3). The concentration and the DOL were quantified by using the calibration curves of both panitumumab at 280 nm (Supplementary Fig. 4a) and the IRDye800 carboxylate at 780 nm (Supplementary Fig. 4b). The presence and amount of residual NHS impurity were determined by the calibration curve of the NHS at 220 nm⁴⁴. QC covers more tests including pH, filter integrity, sterility and bacterial endotoxin levels. These tests ensure that the final product isolated in the three different batches complies with the cGMP standards for human use. Moreover, our group has previously reported data on the stability and safety of a large batch of panitumumab–IRDye800CW⁴⁵, demonstrating that the agent remained safe and stable for administration over a 54-month period for the contract research organization-manufactured material and for a minimum 6-month stable period for the small-batch process. Small-batch production also offers a lower risk option for the stability of the labeled monoclonal antibody, because the doses will be consumed in a short time frame compared to a large-batch option. Although sufficient formulation and storage optimization can help to ensure a long time for expiration, this is too resource intensive for early-phase academic research.

The results shown in Table 3, Entry 10 indicate that using 5.6 equivalents of IRDye800CW NHS, which corresponds to 15 mg per 300 mg of panitumumab (Supplementary Fig. 9), resulted in a D/P ratio of 1.83 ± 0.15. This degree of labeling is approaching the upper limit of the release criteria (1 < D/P ratio < 3). In our PV campaign runs, we adjusted the number of IRDye800 equivalents to 4 by using 10 mg per 300 mg of panitumumab. This adjustment yielded D/P ratios of 1.75, 1.74 and 2.00 in qualification runs 1, 2 and 3, respectively.

The results of the PV campaign presented in Table 6 validate the robustness of this automated approach and ensure the reproducibility of the panitumumab–IRDye800CW quality production that is in compliance with cGMP standards and suitable for human use.

Supplementary Material

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41596-026-01344-y.

Key points.

• cGMP-quality optically labeled monoclonal antibodies (panitumumab–IRDye800CW and nivolumab–IRDye800CW) are produced by using an automated radiosynthesis module with disposable components.

• Single-use components eliminate the need for a dedicated clean room facility while automation simplifies the production and purification process, lowering the cost and time required to produce therapeutic monoclonal antibodies.

Data availability

Data for this Protocol can be found in the supplementary information.