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Emerging Trends in Single-Use Technology in the Manufacturing of Antibody-Drug Conjugates

Single-use technology, designed for the manufacturing of biopharmaceutical products, has made major inroads over the last 30 years. First introduced in the late 1970s in the form of disposable capsules and a range of filters, single-use technologies were revolutionized in the late 2000s with the introduction of single-use 2D and 3D process containers and filter assemblies for mixing and storage systems. Today, these technologies have been adopted across the upstream manufacturing process, downstream purification and fill-finish of entire classes of biologic drugs.

The adoption of single-use technology is especially growing in the development and manufacturing of biologics and complex drugs like Antibody-drug Conjugates (ADCs).

Antibody-drug conjugates
ADCs are highly potent biopharmaceutical drugs designed as a targeted therapy in the treatment of cancer. They are highly hazardous materials, often with occupational exposure limits (OEL) below 100ng/M³/8Hr work day.

The acute potency of ADCs creates a significant risk to personnel involved with the various manufacturing stages. The accepted method to counter the risk of exposure to ADCs is the implementation of so-called barrier isolation systems. These systems are recognized as the highest level of current containment technology, creating both respiratory and dermal protection.

While the use of glass or stainless-steel legacy systems may effectively protect operators, significant equipment decontamination is required. Since most ADCs are produced on a small (production) run/campaign with manufacturing typically taking days rather than months, the cleaning validation burden associated with a hard-shell glass or stainless-steel isolator can be an issue.

Alternative to traditional technology
“Single-use technologies are an alternative to traditional glass or stainless-steel manufacturing with the key difference in their materials of construction. Glass and stainless-steel equipment have decades of historical data and, as a result, their use is well characterized,” noted Karen Green, product manager for single-use assemblies at MilliporeSigma.

“Single-use systems are commonly composed of polymeric materials, which are not as well-known or characterized for biologics processing. These differences result in different approaches to validation and qualification,” she added.


Figure 1.0 A typical ADC process work flow. The input mAb is prepared into reaction conditions by simple dilution or through buffer exchange by ultrafiltration/diafiltration (UF/DF), a very economical, high-yield and robust separation process. In the processing equipment, the antibody will be further modified, then conjugated with the drug linker to form the crude ADC.

Benefits
In the manufacturing of highly complex active pharmaceutical ingredients (APIs) such as ADCs, single-use technologies offer specific benefits in the upstream manufacturing and production of monoclonal antibodies (mAbs) and downstream bioconjugation.

For example, single-use technology enables faster process changeover and facility flexibility that is not possible when traditional equipment is used.

“Since each single-use system is pre-sterilized and used only once, there is no need to sterilize or clean systems between batches, saving time and enabling manufacturers to produce multiple products within the same facility. Furthermore, single-use systems are often mobile, allowing them to be moved within the facility as needed, enabling additional facility flexibility,” explained Mary Robinette, principal project engineer at MilliporeSigma.

Contract development and manufacturing organizations
According to various reports, 70-80% of the manufacturing of ADCs is outsourced to contract development and manufacturing organizations (CDMOs).[1]

“Due to [this] increased outsourcing pattern, CDMOs entertain many different types of ADCs. The use of single-use technology by CDMOs will help speed up the product change over time, avoiding time spent in establishing cleaning methods for each product that is produced, and eliminating upfront investment for expensive capital equipment such as reactors for each product,” said Gang Yao, Ph.D., principal scientist, process & analytical development at MilliporeSigma.

“In the end, customers benefit from lower manufacturing costs and speed to market. The faster turnover will result in more batches made to meet the commercial demand,” he added.

Implementation of single-use technology
Single-use technologies have advanced in several ways over the past decade. Their materials of construction are better known and have established leachables and extractables profiles, and manufacturing techniques have evolved leading to cleaner and more robust films.
Due to these advancements, the adoption rate of single-use has steadily increased across the biopharmaceutical industry, including ADC manufacturing. However, ADC manufacturers will need to be assured of solvent compatibility with bag liners and other single-use components since the manufacturing of ADCs often involves either dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) for the conjugation process.

They also will need to trust that the potential for a leak during the conjugation process is extremely low and that they can successfully scale from a smaller development scale to large-scale GMP production.

Single-use technology suppliers, like MilliporeSigma, have recognized these concerns and have demonstrated that the materials in single-use technologies are indeed compatible with two commonly used solvents (DMSO and DMA) at the temperatures and duration typically used for ADC processing.

Addressing the aggressive conditions used during bioconjugation to ensure compatibility with the polymeric materials used in single-use assemblies and understanding extractables and leachables under these conditions are vital. MilliporeSigma provides supporting data to ensure that the use of solvent during the manufacturing process will not negatively impact the conjugate by demonstrating solvent compatibility as well as sharing representative leachable and extractable data.

MilliporeSigma also has demonstrated that small-scale development batches can be successfully scaled up to large-scale GMP batches using a completely single-use process, guaranteeing operator safety at all steps in the manufacturing. In this single-use process, the fluid contact materials do not change, only the size of the components of the process assemblies. “However, operator safety becomes very critical with the use of more potent linker payloads that typically demonstrate IC50 values in the low-to-mid picomolar range,” Yao added.

Mobius® FlexReady Solution with Smart Flexware™ Assemblies for Chromatography and TFF
Mobius® Mixer
Mobius® Single-use Bioreactors
Pellicon® Capsules with Ultracel® Membrane

Tangential flow filtration
Tangential flow filtration (TFF) is a common unit operation in ADC manufacturing and enables concentration and exchange to pre-formulation buffer.[2]

The presence of toxic linker-payloads following conjugation presents challenges in traditional TFF operations. The scale of TFF also can be a challenge.

“MilliporeSigma has developed a completely enclosed single-use TFF capsule. This device is shipped gamma sterilized with RO (reverse osmosis) water, which reduces flushing requirements and enables faster batch turnaround while utilizing the same Ultracel 30 kDa membrane found in our traditional flat-sheet devices,” noted Nicholas Landry, group product manager ultrafiltration at MilliporeSigma.

“The device was engineered with operator safety and containment in ADC processes as design principles,” he added.

Upstream and downstream processing
While single-use technology has generally been used in upstream processing in the manufacturing of mAbs, the technology is now also available in downstream bioconjugation.

One of the major benefits of single-use technology in downstream processing is bioburden control. Single-use technology offers a more closed processing opportunity compared to traditional glass or stainless-steel reactors, thus reducing the opportunities for bioburden growth.

Another significant benefit of single-use technology is that there is no cross-contamination from inefficient cleaning, allowing faster turnover between process changeovers in a biopharmaceutical manufacturing facility, while at the same time, reducing cleaning validation requirements.

In the final verdict, single-use technology has proven, compared to traditional methods, to be a flexible, cost-effective and efficient alternative that provides improved safety. There is no cross-contamination from inefficient cleaning and no cleaning required between batches, resulting in a quicker turnover of the facility.

Reference
[1] Roots Analysis, Antibody Drug Conjugates Market (2nd Edition), 2014 – 2024.
[2] Czapkowski B, Steen J, et al. “Trial of High Efficiency TFF Capsule Prototype for ADC Purification,” ADC Review, April 12, 2017. [Article]


Last Editorial Review: November 20, 2018

Featured Image: Scientists in Laboratory. Courtesy: © 2010 – 2018 Fotolia. Used with permission.

Copyright © 2018 InPress Media Group. All rights reserved. Republication or redistribution of InPress Media Group content, including by framing or similar means, is expressly prohibited without the prior written consent of InPress Media Group. InPress Media Group shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. ADC Review / Journal of Antibody-drug Conjugates is a registered trademarks and trademarks of InPress Media Group around the world.

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Trial of High Efficiency TFF Capsule Prototype for ADC Purification

Antibody-drug conjugates (ADCs) are an emerging class of highly targeted cancer therapies in which a monoclonal antibody is chemically conjugated to a cytotoxic drug (payload). These complex biochemical moieties are comprised of three essential components: the monoclonal antibody, the payload, and the

Photo 1.0. Brooke Czapkowski (corresponding author) holding the TFF capsule 0.11 m2 prototype.

linker, which holds the moiety together. Upon targeted recognition of the specific cancer cell receptors, the antibody becomes internalized by the cell, which makes it an effective vehicle for therapy. Once the ADC has been internalized, the cytotoxic drug is released, enabling it to kill the cancerous cell. Common mechanisms of action for these drugs include microtubule inhibition and DNA damage.

Due to the toxic nature of ADC compounds, non-toxic linker payloads (“mimics”) have been designed in order to study and effectively model ADCs. ADC mimics are comparable in the basic structure to the actual cytotoxic ADC, and can be conjugated, purified, and filtered just as normal, cytotoxic ADCs would.

These conjugation reactions require organic solvents and excess equivalents of linker payload (either cytotoxic or mimic). Solvent and excess free drug or mimic are much smaller molecules than the conjugate and can be removed rapidly from the drug substance by diafiltration with tangential flow filtration (TFF).

 The TFF step presents a safety concern to operators due to the high toxicity of the payload and the open cassette format of traditional TFF devices. TFF cassettes only seal when installed in a compression holder, thus leaving a risk of operator exposure to process fluid after filter removal for storage or disposal. A new TFF device format in development comprises a self-enclosed, pre-sterilized capsule that does not require a holder, thus dramatically improving the ease of use and safety of the TFF operation. The new device also provides high efficiency comparable to TFF cassettes, allowing for use of similar pumps and membrane areas. This simplifies conversion between the two formats for process development and production.

A proprietary, purified monoclonal antibody (~150 kD) was chemically conjugated to a proprietary linker-payload mimic molecule (~1 kD) to make an ADC mimic, and this mimic was used to compare the prototype TFF capsule to state of the art benchmark cassettes. Dimethyl sulfoxide (DMSO) clearance, ADC yield, aggregate level, and flux were analyzed in order to understand the comparability between these two TFF devices. Safety and efficiency of the two devices were also evaluated.

Materials and Methods
Four devices were tested over a two-day period: two Pellicon® 3 0.11 m2 Ultracel® 30 kD nominal molecular weight cut-off cassettes (MilliporeSigma, Bedford, MA) and two TFF capsule 0.11 m2 prototypes also with 30 kD Ultracel® membrane (MilliporeSigma; See Photo 1 | Figure 1). One capsule and one cassette were run the first day on parallel TFF systems and the second capsule and cassette were run in a similar fashion on the second day. The TFF process flow diagram can be found in Figure 1.

Comparability Data
Key device and system characteristics are shown in Table 1. All values are determined to be within the acceptable range except the hold-up for the cassette system on Day 1, which appears to be in error since tubing was retained for Day 2, where system values are much lower. System hold-up volume was determined by the change in the fully retained protein concentration upon addition of the known stock solution.

It is likely there was an error in pulling the diluted concentration sample from the system or in the UV measurement; therefore, the cassette hold-up value for Day 1 was omitted from the average (Table 1).

DMSO was efficiently cleared from the ADC mimic feed solution by both devices (Figure 3). Ten diafiltration wash volumes reduced DMSO concentration by a factor of over 10,000 (4 log reduction) with a sieving coefficient (S) of 1, which is ideal (Fig 3B). DMSO concentration was reduced by a total factor of around 100,000 (5 log reduction) or more after 17-20 diafiltration volumes.

The filtrate flow rate was a function of transmembrane pressure (TMP) below 15 psi for the capsule and below 20 psi for the cassette, due to the capsule’s higher permeability (Figure 4). Overall, the fluxes were stable throughout the diafiltration step for both devices. The TMP was set to 15 psi for the capsule; the cassette started at 10 psi, by manual error, and then was corrected to the target 15 psi after 3 diafiltration volumes (Figure 5A). Both formats could obtain a maximum flux of about 90 LMH, L/m2-hr (Figure 5B).

Yields were comparable and acceptably high for the two device formats (Table 2), and the aggregate formation rate for the ADC mimic was also similar (Figure 6).

A summary of the performance comparability of the two devices based on the target parameters studied is shown in Table 3. The capsule prototype demonstrated comparable performance to the cassette when run at the same feed flow rate and TMP.

Applicability Data
The capsule was user-friendly as summarized in Table 4. The capsule was lighter, more mobile, and easier to set-up than the cassette since it did not require a holder (5kg) and had fewer connections to establish the flow paths. Approximately 45 minutes was saved per alpha trial run by the capsule because the sanitization step was not needed. The potential time saved on the GMP manufacturing floor can be many times higher. The capsule fit well within the typical TFF system flow path used for cassettes, and was also safer for the operator, as it did not need to be opened during disassembly to remove the filter: the capsule is self-contained.

Conclusion
An ADC mimic was used to compare the prototype TFF capsule to state-of-the-art benchmark cassettes. Both devices demonstrated similar process performance and clearance of the organic solvent (DMSO). Based on the assessment of the two device formats, the capsule could be a safer, more time-efficient device for ultrafiltration/diafiltration processes of antibody drug conjugates. Follow up studies will be conducted to evaluate scalability of different sizes and types of devices using actual cytotoxic ADC material.


Brooke Czapkowski (1), Jonathan Steen (2), Eric Bortell (1), Vimal Patel (1), Ye Joon Seo (1), Jim Jiang (1), Julius Lagliva (1), Deanna Di Grandi (1), and Mikhail Kozlov (2)

(1) Pfizer, Pearl River, NY; (2) MilliporeSigma, Bedford, MA


March 27, 2017 | Corresponding Authors: Brooke Czapkowski and Jonathan Steen | DOI: 10.14229/jadc.2017.11.04.001

Received: March 27, 2017 | Accepted for Publication: April 10, 2017 | Published online April 12, 2017 |

Last Editorial Review: April 11, 2017

Featured Image: Chemical Glass. Courtesy: © Fotolia. Used with permission.

Creative Commons License
This work is published by InPress Media Group, LLC (Trial of High Efficiency TFF Capsule Prototype for ADC Purification) and is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Non-commercial uses of the work are permitted without any further permission from InPress Media Group, LLC, provided the work is properly attributed. Permissions beyond the scope of this license may be available at adcreview.com/about-us/permission.


Copyright © 2017 InPress Media Group. All rights reserved. Republication or redistribution of InPress Media Group content, including by framing or similar means, is expressly prohibited without the prior written consent of InPress Media Group. InPress Media Group shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. ADC Review / Journal of Antibody-drug Conjugates is a registered trademarks and trademarks of InPress Media Group around the world.

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