ADC 生产用一次性系统中的可提取物研究：ReadyToProcess™ 层析柱外壳与 ÄKTA ready™/readyflux™ 一次性流路在 DMA、DMSO 中的提取研究

Single‑use (SU) systems have great potential for use in antibody‑drug conjugate (ADC) manufacturing. The use of organic solvents in the ADC process might, however, raise questions about potential leachables from the plastic and elastomeric materials of single‑use components. To address those concerns, we performed extractables studies on a disposable chromatography column housing and two different disposable flow paths. The extractables studies were performed with two solvents commonly used in the ADC cytotoxin conjugation step: DMA and DMSO.

The studies were designed to ensure that conditions were exaggerated compared with existing ADC manufacturing processes. Extractable organic compounds and trace elements from the single‑use components were identified and semi‑quantitated with a complementary set of analytical techniques. The low levels of extractables found in this study support the use of ReadyToProcess™ columns, ÄKTA readyflux™ flow kits and ÄKTA ready™ flow kits in ADC processes.

Introduction

ADCs are biotherapeutic molecules consisting of a cytotoxin coupled to a monoclonal antibody (mAb) by a linker. The target specificity of the mAb enables delivery of the toxic drug to cancer cells, while minimizing collateral damage to healthy cells. mAbs used in ADC production are typically manufactured according to traditional processes, including purification via protein A‑platform processes (1, 2). Before coupling the linker, the mAb needs to be transferred to a suitable solution. This solvent exchange is normally performed by an ultrafiltration/diafiltration (UF/DF) operation. After the linker coupling reaction, the next step is the conjugation reaction, which couples the cytotoxic drug. Figure 1 provides an example workflow for manufacturing an ADC from a bulk mAb product. Conjugation reactions are typically performed in a solvent containing either N,N‑dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) (3) at concentrations from 10% to 15%.

Fig 1. Simple workflow for preparing an ADC from bulk mAb.

SU systems are well suited to ADC manufacturing for several reasons. Importantly, SU systems minimize operator exposure to toxins, while also protecting the product from the operator and environment. The high potency of ADCs means that relatively small amounts of products need to be produced per batch. The small batch sizes are well suited for incorporation of SU technology, which provides a cost‑efficient solution for multi‑product manufacturing. In addition to the lower upfront capital cost compared with reusable systems, SU technologies are quicker to start using because they are supplied ready to use. Cleaning and cleaning validation between manufacturing campaigns is unnecessary and the risk of carryover of cytotoxin from one batch to another is minimized. Because cleaning is not performed, SU technologies minimize the volume of contaminated waste that must be handled and disposed of.

To support the adoption of SU technologies in ADC production, relevant extractables information is needed. Therefore, extractables studies were performed on three SU products: the ReadyToProcess™ 1 L column housing, disposable ÄKTA ready™ low flow kit for chromatographic systems and ÄKTA readyflux™ flow kit for tangential flow filtration (Fig 2 and 3).

Fig 2. ÄKTA ready™ system with a 20 L ReadyToProcess™ column and ÄKTA ready™ flow kit. In this study, 1 L column housings were used.
 

Fig 3. ÄKTA readyflux™ tangential flow filtration system with flow kit.

Materials and methods

The goal of the extraction studies was to characterize extractables profiles with equipment and conditions relevant to current ADC manufacturing processes. The studies were designed and performed with advice from customers who use disposables in their ADC processes.

Read about the study design, process, methods and analysis here

Results and discussion

ÄKTA readyflux™ flow kit

In the extract from the ÄKTA readyflux™ flow kit incubation, five organic compounds were found above reporting limit for each solvent (30% DMSO and 30% DMA). All compounds identities were assigned confirmed and the majority of them were related to antioxidants and silicone material. Some differences between the solvents could be seen, but the total amount of extractables found did not differ significantly. The compound with the highest abundance was a carboxylic acid, semi-quantified to 430 µg per ÄKTA readyflux™ flow kit.

For ICP-SFMS, the results were aligned between the solvents: four elements were above the reporting limit, the same elements in both solvents, with potassium having the highest abundance (3.8 mg/flow kit in 30% DMA).

ReadyToProcess™ column housings

No organic extractable compounds were found above the reporting limit with HS‑GC‑MS or GC‑MS in the extraction samples with 15% DMA or 15% DMSO. The results with LC‑MS showed one organic compound that was present in both 15% DMA and 15% DMSO. The extractable compound was assigned a confirmed identity level as an identified compound related to a curing agent used with elastomeric material. The concentration was estimated below 200 µg/L (ppb) in both extraction solvents (that is, less than 200 µg/ReadyToProcess™ column).

Analysis with ICP‑SFMS showed low levels of a few extractable elements. The most abundant were calcium (< 100 µg/L) and magnesium (< 25 µg/L), followed by zinc (< 10 µg/L) and barium (< 2 µg/L). The extractable elements were found at a similar level in both 15% DMA and 15% DMSO.

ÄKTA ready™ low flow kit

The results showed five organic extractable compounds above the reporting limit with HS‑GC‑MS, GC‑MS and LC‑MS. Two of the compounds were found in 15% DMA and three compounds were found in 15% DMSO. All five extractable compounds were assigned a confirmed identity level as identified compounds related to polyamide and silicone materials and one solvent. The highest abundant extractable compound was present at a concentration below 600 µg/L (that is, 410 µg/ ÄKTA ready™ low flow kit, because the extraction volume was 700 mL).

Analysis with ICP‑SFMS showed that the most abundant extractable element was silicon, which was present below 9 mg/L (ppm). Additionally, calcium, barium, zinc and copper were found at lower levels. The extractable elements were found at a similar level in both 15% DMA and 15% DMSO.

Assessment of results

A general toxicity and safety evaluation of extractable compounds as a worst case was conducted. The evaluation can only be general, because the specific details regarding the route of administration, dosage level, or toxicity of the proposed drug compounds will differ between different ADCs.

Toxicological information and a derived risk index (RI) for eight out of the ten identified extractable compounds were listed in a reference containing compiled safety impact information (4). In that reference, risk indices were obtained by subjecting toxicological safety data such as no observed effect levels (NOELs), no observed adverse effect levels (NOAELs), lowest published toxic dose (TDLOs) and others to a systematic evaluation process using appropriate uncertainty factors. An RI value represents a daily intake value for life‑long intravenous administration. Two additional RI values were derived for the final identified extractable compounds from a reported NOAEL value and appropriate uncertainty factors.

Assumptions for the assessment were:

• Single‑use equipment included in this assessment comprise the column housing, the chromatography flow kit and the tangential flow filtration flow kit. All extractables from these disposables end up as impurity in the ADC product.
• Batch size is 5 g, considered a small batch, representing a worst case.
• Dosage is 3.6 mg/kg given every three weeks (21 d cycle), that is, 3.6 mg/kg × 70 kg bodyweight (bw)/21 d = 12 mg/person/d.

Potential exposure to extractables was calculated from the highest result on extractables divided with the batch size of ADC multiplied with the daily dosage of ADC (Table 6).

Table 6. Calculations for the safety assessment of the eight extractable compounds (highest concentration chosen if reported more than once)

¹ÄKTA readyflux™ flow kit
²ReadyToProcess™ column
³ÄKTA ready™ flow kit

Assessment of the results according to the listed assumptions shows that the potential exposure to extractables is well below the RI for each extractable compound. Therefore, extractables from the ÄKTA readyflux™ flow kit, ReadyToProcess™ column housing and ÄKTA ready™ flow kit should pose no safety concern for use in ADC manufacturing within the conditions of this study.

Conclusions

The low levels of extractables found in this study demonstrate the chemical compatibility of ÄKTA readyflux™ flow kits, ReadyToProcess™ columns and ÄKTA ready™ flow kits with two organic solvents typically employed in ADC manufacturing processes: DMSO and DMA. Detailed results of these studies are available in the validation guides for these SU components to supplement existing data generated with aqueous solvents and ethanol. Along with other SU components and systems, the ÄKTA readyflux™ flow kits, ReadyToProcess™ columns and ÄKTA ready™ flow kits offer a solution to some of the main challenges in ADC manufacturing.

REFERENCES

1. Application note: Three‑step monoclonal antibody purification processes using modern chromatography media, Cytiva, CY136545-21May20-AN;2020.
2. White paper: Purification of monoclonal antibodies using modern chromatography media and membranes. Cytiva, CY13545-19May20-WP;2020.
3. Ding W. Risk‑based scientific approach for determination of extractables/leachables from biomanufacturing of antibody–drug conjugates (ADCs). Methods Mol Biol. 2013;1045:303–311. doi: 10.1007/978-1-62703-541-5_20.
4. Jenke D., Carlson T. A compilation of safety impact information for extractables associated with materials used in pharmaceutical packaging, delivery, administration and manufacturing systems. PDA J Pharm Sci and Tech. 2014;68:407–455. doi: 10.5731/pdajpst.2014.00995.