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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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California Life Sciences Association Rewards Sutro Biopharma with Outstanding Partner Award

This week, during the California Life Sciences Association (CLSA) annual Pantheon Ceremony (DiNA Awards), Sutro Biopharma received the Outstanding Partner Award. By giving this award to Sutro, the CLSA recognized the company for its life science achievements throughout 2015.

The recognition of Sutro Biopharma is, in part, based on the development of novel mono- and bi-specific antibody-drug conjugates or ADCs using the company’s cell-free biologics development platforms, Xpress CF and Xpress CF+.

In addition, Sutro’s ADC technology, which allows researchers to incorporate non-natural amino acids at any site in an antibody structure, has been developed to allow loading of an antibody with multiple different agents and to enable a potential higher uptake of the drug in the tumor cell through improved stability of antibody-drug conjugates.

Photo 1.0. William Newell, chief executive officer of Sutro Biopharma: "This award recognizes our partnerships as first-rate collaborations designed to achieve the best for patients and health care providers."
Photo 1.0. William Newell, chief executive officer of Sutro Biopharma: “This award recognizes our partnerships as first-rate collaborations designed to achieve the best for patients and health care providers.”

Collaboration
In October 2014, Sutro entered into a broad strategic collaboration and option agreement with Celgene Corporation to discover and develop multi-specific antibodies and antibody-drug conjugates. The scope of the agreement also included identification of novel targets, such as PD-1 and PD-L1.

The same year Sutro entered a multi-target strategic collaboration with EMD Serono (part of Merck KGaA; Darmstadt, Germany) to discover and develop novel ADCs. This collaboration allows the biopharmaceutical division of Merck KGaA to take advantage of Sutro’s technology platforms in its oncology programs to develop ADCs for multiple undisclosed targets.

ADCs have the potential for directly targeting cancer cells while safeguarding healthy tissue.  Using this technology, researchers at Sutro are combining Merck KGaA’s knowledge about target biology with their own technological and discovery capabilities to jointly develop novel ADCs.

In 2014, at the time of concluding the agreement, Andree Blaukat, Senior Vice President and Head of Translational Innovation Platform Oncology at Merck Serono, the biopharmaceutical division of Merck KGaA, Darmstadt, Germany noted:  “We continue to explore opportunities that will allow us to better understand the potential ADCs have in directly targeting cancer cells and our collaboration with Sutro is reflective of our ongoing commitment to advancing innovation that may provide new therapies for patients.”

Strategic Approach
By creating strategic partnerships, Sutro aims to advance the company’s position as a leading drug discovery partner to leading pharmaceutical companies as well as develop drug candidates that may ultimately address the unmet medical needs of patients as.

These unique partnerships allow researchers at Sutro to advance their efforts to develop antibody therapeutics, engineered to deliver a cytotoxic agent to cancer cells. The Outstanding Partner Award bestowed by the California Life Sciences Association confirms the validity of Sutro strategic partnerships approach.

“We are honored to pioneer a new generation of cancer therapeutics with our partners Celgene and EMD Serono,” noted William Newell, chief executive officer of Sutro Biopharma. “This award recognizes our partnerships as first-rate collaborations designed to achieve the best for patients and health care providers. Our platform and product development efforts bear enormous promise. These collaborations have worked to accelerate the development of oncology therapeutics, with compelling patient benefit.”

Photo 2.0. Getting the grand tour of the new manufacturing facility from Sutro VP Henry Heinsohn – Also in the photo William J. Newell, Congresswoman Jackie Speier and Ben Cohn, district representative for Assemblyman Kevin Mullin.

Cell-free biologics development platforms
Xpress CF and Xpress CF+, a biochemical synthesis system that enables rapid and systematic evaluation of protein structure-activity relationships, as well as rapid and predictable scalability for manufacturing in Sutro’s cGMP facility, are key elements to its strategic collaborations, driving forward the development of next generation immuno-oncology therapeutics.

At the end of October, to help advance the company’s cutting-edge manufacturing capability, Sutro unveiled the only cGMP cell-free manufacturing facility in the world. In this new facility, the company will be able to produce highly their specialized and selective protein based cancer therapeutics more quickly, efficiently and in greater amounts than before.

A Blossoming Community
The California Life Sciences Association annual Pantheon Ceremony shows the strength and the importance of the pharmaceutical industry for the state. It also showcases the solutions innovators like Sutro are developing to meet the medical needs of patients.

“California’s life sciences community continues to blossom and grow to new heights thanks to our statewide innovators, entrepreneurs and executives,” noted Sara Radcliffe, President & CEO of California Life Sciences Association (CLSA). “The high caliber of this year’s awardees is a testament to the strength of our sector, and a promise of big things to come. With 2,848 life sciences companies employing nearly 300,000 people all across the state, California’s life sciences community is an increasingly important driver of our economy and the source of promising innovations for patients and consumers around the world,” she continued.


Last Editorial Review: November 18, 2015/Last update: November 23, 2015

Photo 1.0: William Newell, Chief Executive Officer of Sutro Biopharma. Photo 2.0: Getting the grand tour of the new manufacturing facility from Sutro VP Henry Heinsohn. Courtesy Photo 1 & 2: © Sutro Biopharma. Used with permission. Featured image: Scientist researching in laboratory, pipetting cell culture medium samples in laminar flow. Courtesy: Fotolia.

Copyright © 2015 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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CPhI Worldwide 2015, Antibody-drug Conjugates and Market Progression

Ahead of CPhI Worldwide 2015, CPhI expert panelist, Vivek Sharma, CEO of Pharma Solutions at Piramal, analyses the challenges in developing and manufacturing of Antibody-drug Conjugates (ADCs) and how the market has progressed over the past few years.


Major challenges faced by companies looking to enter the ADCs market are the availability of technologies, which are few as yet, and the complex supply chain to manage while going for the clinics. Many a time a sponsor ends up working with 4 different CMOs before the clinical supplies are available for dosing – one each for antibody, cytotoxic molecules, the ADC conjugate & the Fill/Finish. Add to it the availability of quality CMOs in the space and this becomes a major stumbling block for the ADC player.

Most high potent facilities are not suitably designed to produce biologics (i.e. environmental standards, particularly microbiological controls may be inadequate) and, similarly, most biologics facilities don’t have high potent capabilities – so it is rare to find a recognized, credible manufacturer that operates in both fields.

Hugely under resourced
As such, despite the increase in development targets for ADCs, the global contract manufacturing sector still remains hugely under resourced, with only a couple of players having the experience & necessary regulatory approvals to commercialize these products.

Piramal is one of the longest established ADC producers (beginning in 2004) and the main challenge when they transitioned was moving from the high potent field to biologics and combining the two practices for successful conjugation. For many CMOs entering this space now, the real issue is building a facility with high containment in mind. Furthermore, having the desired skillsets to work efficiently with ADCs is crucial, as each new ADC requires specialist adoption and a track record of prior experience is understandably what all pharma companies are searching for in a partner.

ADC manufacturing involves a complex supply chain and it is a major concern for pharma clients, as they seek to de-risk the process as much as possible. So, our view is that you will see pharma consolidating a majority of their projects with a few specialized players.

Linker chemistry improvements
Looking at the industry as a whole, over the past few years, we have definitely seen improvement over the aggregation of proteins. In the past, first generation drug linkers would produce 20-30% aggregation of proteins, which is a big problem as it gives a low yield. However, through linker chemistry improvements this has now dropped to 1-2% of aggregation, which is a giant step forward. The next stage is a consolidation amongst specialists.

Supply chain
The complexity of the supply chain and market demand for looking at projects more in their entirety, from production of mAb all the way through to fill/finish; the more parts of these that can be aligned, the easier it is for all parties. There is definitely a distinct benefit for the client, particularly if the different supply chain areas are across the same company – with shared project management, established point of contacts, and using the same quality and release systems.

Some of the industry’s most active producers of antibody-drug conjugates – Almac, Calalent, Lonza and Piramal – will be in attendance at CPhI Worldwide 2015.


Last Editorial Review: October 2, 2015

Feature Image: IFEMA – Feria de Madrid, Madrid, Spain. Feature image Courtesy: © IFEMA – Feria de Madrid, Madrid, Spain. This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. CC BY-SA 3.0

Copyright © 2015 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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Event Preview: CPhI Worldwide 2015

CPhI Worldwide 2015 returns to Madrid, Spain, for the 26th edition, bringing with it the booming pharmaceutical industry ready for a plethora of networking, learning and sharing opportunities

The pharmaceutical ingredients, outsourcing, packaging and machinery sectors continue to grow apace and CPhI attendees are hugely positive about industry and new business prospects over the coming 12 months and beyond. CPhI Worldwide provides the initial platform to spark innovations, bringing pharma suppliers together to meet and develop new dynamic approaches to industry challenges.

CPhI has progressively been adding to its portfolio with thought leadership through its Pharma Forum and Pre-Connect Congress. Also, the eagerly anticipated and expanded, CPhI Pharma Awards, provides recognition on a global scale to pharma’s most transformational companies, products and services.

Following the success of last year’s 25th Anniversary event, 2015 promises to be another record-breaking show, with over 36,000 attendees and some 2,500 exhibitors from over 150 countries expected at IFEMA (Feria de Madrid, Spain).

Industry Growth
During the last few years, the event has mirrored the growth of the industry, and remains integral to the pharmaceutical community agenda, and offers engaging, world-class content. CPhI Worldwide brings together the leading suppliers, buyers and experts for ingredients, APIs and excipients globally, with ICSE connecting international outsourcing solution providers with the pharmaceutical community.

Running alongside the pharmaceutical ingredients and outsourcing halls are further sister brands Innopack and P-MEC, allowing visitors to quickly identify the right hall for their needs, spanning the entire pharmaceutical supply chain. InnoPack brings together buyers and specifiers from the packaging and pharmaceutical industries. P-MEC Europe features exhibitors from traditional large-scale capital equipment to companies focussed on instrumental analysis, measuring and testing technologies, materials testing, quality control and laboratory.

More than a meeting platform
However, CPhI Worldwide is now much more than a meeting platform – for 2015, the show floor has been expanded, not only for the increased number of attendees and exhibitors, but also to accommodate new innovations brought to the show. This year sees the introduction of the CPhI Pharma Forum – a dedicated content village that will provide a central hub to examine thought leadership from media partners and the CPhI Pharma Insights Reports. The Pharma Forum will also be the location for the exhibitor and visitor party, and will include exhibitor Innovation Galleries, the CPhI Pharma Awards and the Pharma Insight Innovation Briefings – offering impartial, in-depth sessions on regional updates and specialist topics covering regulation, QC, traceability, sustainability and health to name but a few.

Another innovation for 2015 are the revamped CPhI Pharma Awards, which now feature five new categories to allow for a wider range of recognition across the pharma industry. Also returning are the free sessions in Speaker’s Corner, which give exhibitors a forum to deliver first-hand presentations to senior pharma attendees from across the globe. To help attendees tailor their time at the show, CPhI Worldwide Global Meetings is a custom matchmaking programme that enables attendees to find and connect with specific companies tailored to their business needs.

CPhI Pre-Connect Congress
Opening the day prior to the main exhibition the CPhI Pre-Connect Congress (12th October), affords senior executives and influential speakers the opportunity to discuss the latest innovations, trends and market developments from across the industry in a series of market-led educational modules. It is organised along two main tracks, with sessions in track one including ‘Formulation & Drug Delivery’; ‘Biologics, Biosimilars & Biobetters’; and ‘API Sourcing & Manufacture’. Track two features modules across ‘generics’; ‘Pharmaceutical Packaging’; and ‘Mergers and Acquisitions’.

Some of the industry’s most active ADC producers – Almac, Calalent, Lonza and Piramal – will be in attendance at CPhI Worldwide 2015. 


Last Editorial Review: October 2, 2015

Feature Image: IFEMA – Feria de Madrid, Madrid, Spain. Feature image Courtesy: © IFEMA – Feria de Madrid, Madrid, Spain. This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. CC BY-SA 3.0

Copyright © 2015 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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Downstream Processing Considerations for Antibody Variant Therapeutics

Abstract
Novel platforms such as antibody derivatives, peptide based therapies, gene and stem cell based therapies are gaining foothold in the market for several reasons, including the need for better Pharmacokinetics (PK)/ Pharmacodynamics (PD), improved potency against disease targets, ability to treat more than one aspect of a disease simultaneously, better and cheaper production processes, reduced side effects and the biosimilar cliff.

In this article, we will focus on 3 types of antibody derivatives- namely Bispecific Antibodies (BsAbs), antibody fragments (Fabs), and fusion proteins. We will include an overview of each and discuss the typical downstream processes, highlighting specific process challenges. Scale up considerations will also be included.


1.0 Introduction
Monoclonal antibodies (MAbs) continue to dominate in terms of the class of therapeutics for the biotechnology industry. However, the overall trend in the biotherapeutics includes transitioning towards molecules that have higher value and improved bioavailability. Traditional Mabs are altered to achieve this goal, and antibody variants such as antibody fragments (Fabs), bispecific monoclonal antibodies (BsAbs), and fusion proteins are being explored.

The term Fab Antibody fragment is self explanatory- it is the Fab fragment from the variable region of an antibody [1]. A bispecific monoclonal antibody (BsAb) is composed of fragments of two different monoclonal antibodies that bind to two different types of antigens [2]. Fusion proteins are produced from gene fusion techniques that allow the production of recombinant proteins featuring the combined characteristics of the parental products [3].

In terms of common expression platforms, Fabs can be expressed in both mammalian and bacterial expression systems. Bacterial expression system is more common for Fabs and they can be expressed in E.Coli as either inclusion bodies or soluble (soluble being more common). BsAbs and fusion proteins are more typically expressed in mammalian cell cultures.

A typical downstream process for these antibody variants consists of

 

 

Fig 1: Typical Downstream Process (click on image to enlarge). Bioreactor/Fermentor > Harvest/Lysis (if bacterial)/Clarification > Capture > Polishing > Virus Clearance (if mammalian cell line) > UF/DF > Final Sterile.

In this article, we outline some of the unique requirements and challenges posed by these antibody variants in terms of recovery, purification, and scale-up/process transfer.


2.0 Part 1 – Recovery

 

 

 

Fig 2: Recovery: Bioreactor/ Fermentor > Harvest / Lysis (if bacterial) / Clarification (click on image to enlarge)

The vast majority of the current therapeutic antibodies including BsAbs and fusion proteins are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to non-human glycosylation patterns [1]. However, Fabs are more commonly produced in bacterial (E. Coli) due to their smaller size and economic considerations. Bispecific antibodies without any glycosylation could be successfully produced in bacteria as well.

For mammalian cell cultures (used for BsAbs and fusion proteins), normal flow depth filtration can be used for primary and secondary clarification steps for process volumes ≤ 2,000L. Depth filtration has also been shown to assist with removal of impurities such as HCP and DNA and improve downstream filter and column capacities. As titers and cell densities increase, the use of agents such as flocculation polymers/ acid precipitation are becoming more common at harvest.

For bacterial expression systems, clarification is often one of the most challenging steps. For soluble proteins, microfiltration- tangential flow filtration (MF-TFF) is often used instead of normal flow filtration. However, centrifugation followed by normal flow filtration (NFF) can be evaluated. Typically the TFF yields higher product recovery and is more economical. There is also a re-newed interest in older technologies such as using DE as a body feed for clarification. With secreted proteins, whole cells are separated from the fermentation broth and the particle size is thus larger; as a result, microfiltration (using TFF), centrifugation and normal flow filtration are all viable options. Endonuclease agents can also be used prior to clarification to digest DNA and RNA and to aid in the efficiency of the clarification process [2].

For sterile filtration post clarification, the capacity is influenced by the bioreactor/fermentor media components. Symmetric PVDF membranes are better suited for the sterile filtration of PEG and hydrolysate-containing media types. Asymmetric PES membranes are also available and can be evaluated. Sterile filtration step should be optimized with respect to product recovery, capacity and operating flux.


3.0 Part 2 – Purification

 

 

Fig 3. Purification: Capture > Polishing > Virus Clearance (if mammalian cell line) > UF/DF > Final Sterile (click on image to enlarge)

Protein A, followed by cation exchange and anion exchange, can be successfully used for the purification of BsAbs, fusion proteins, or Fabs containing the Fc region. For molecules that do not contain an Fc region, capture is typically achieved using cation exchangers or mixed mode resins in bind/elute mode depending on the molecular characteristics of the target protein. A subsequent polishing step for improving the resolution generally follows the capture step. This polishing step could be ion exchange (IEX) or hydrophobic interaction chromatography (HIC) depending on the previous step. And sometimes a third chromatographic step is required, depending on the separation results from the previous steps. In addition to resins, membrane adsorbers are also used for the polishing steps in (usually) a flow-through mode.

Virus filtration is not needed for bacterial expression systems due to the absence of adventitious viruses. For the proteins expressed in mammalian cell cultures, demonstrating viral clearance is a regulatory requirement. Some fusion proteins or BsAbs can be similar in size to virus filter membrane pore sizes (20nm), leading to significant process challenges in terms of filter capacities and flux rates. For these types of molecules, asymmetric PES parvovirus filter should be evaluated first, with and without prefiltration. If product recovery is an issue, regulatory agencies have accepted the use of non-parvovirus filters [2]. For smaller molecules, asymmetric PES parvovirus filters are recommended. Membrane-based prefilters could be used to normalize the feed (with respect to aggregate and impurity levels), increase capacity and reduce operating costs for this process step [2]. Special care should be taken when outlining the virus validation step as this will dictate achievable process loadings [2].

For the ultrafiltration/ diafiltration step, vendors typically recommend using filters 3-5X tighter than the molecular weight of the target molecule. Therefore, for Fab molecules that are typically small in comparison to Mabs (approximately 10kD – 80kD), 1-10 kD molecular weight cut-offs tangential flow devices are commonly used for the ultrafiltration/ diafiltration (UF/DF) step. As a result, the permeate fluxes can be lower. For BsAbs, the typical UF/ DF filters range from 30-50kD MWCO.

Additionally, some molecules can be PEG-ylated to improve bioavailability, which leads to higher viscosities as concentration increases. Also, because of the interest in subcutaneous applications, the target molecules are being concentrated to higher concentrations. For these reasons, the influence of the type of screen (screens help create turbulence and promote mass transfer) in flat sheet devices is paramount and should be taken into account in small scale optimization studies (1). A final consideration for E. coli expressed Fab molecules is downstream endotoxin removal. This is often achieved through anionic membrane adsorbers or charged membrane filters [1].

For the final sterile filtration, asymmetric PES membranes offer higher fluxes and capacities than symmetric membranes- however both PES and PVDF membranes should be evaluated at this stage. Attention should be paid to the final sterile filtration step as well, especially with respect to product recovery.


4.0 Part 3 – Scale Up and Process Transfer Considerations

Factors to consider when scaling-up current antibody and antibody variant processes depend on the stage and goals for the project, including the molecule’s pre-clinical or clinical phase, speed to market, process economics, manufacturing and operation flexibility, expertise, facility infrastructure, and batch volumes. Pros and cons of these factors can be weighted to decide how to proceed with the scale up logistics. In some cases, companies may lean towards utilizing single-use, stainless steel, or a hybrid for the manufacturing process. Additional considerations include building or using an existing facility, or outsourcing manufacturing to take advantage of the PD experience and infrastructure from contract manufacturing organizations (CMO).

Single-use processes have the inherent “out of the box, and ready to use” benefits, easing implementation.   With single use processes, users benefit from a lower investment in the upfront capital in comparison to a fixed facility with stainless steel systems, specific infrastructure requirements, such as steam and CIP/SIP, are likely not needed, and validation is minimal and/or eliminated [4]. For a multi-product facility, and in cases where batch volumes may vary and be less than 2000L, the additional benefits of a single-use approach may come from the quicker turnaround times from batch to batch, lower risk of cross product contamination, flexible volume manufacturing, and overall economics and facility fit. All of these factors contribute to the delivery of a process with speed to market needs, improved economics, and process flexibility.

A stainless steel facility can be considered for late stage molecules, multiple and large campaigns, and batch volumes greater than ~2000L where single-use systems may be a limitation.   In this case, the facility and equipment implementation would have a larger capital cost and initial validation investment; however, the long-term utilization of these assets can bring a return of investment and pay for itself over time, depreciation of equipment is incorporated, and other factors of a long term and multiuse facility may make economics more feasible [4]. A manufacturing facility may also incorporate a hybrid of single-use and stainless steel infrastructure to accommodate all needs of the project’s stage and goals. CMOs are well equipped with both types of facilities and process expertise, which may be more appealing in cases where there is a facility throughput limitation and/or speed to market may require outsourcing.

In addition to specific facility needs, each unit operation has its own specific rules for scale-up. In some cases, linear scalability can be accomplished for some technologies such as filtration; however, system scale-up is sometimes overlooked and can be the cause for deviation or unexpected process performance [5]. Fluid dynamics, hold-up volumes, frictional losses, hardware requirements and yield recoveries are factors to strongly investigate prior to scaling up. Ultimately, thorough process transfer studies must be completed to ensure the process meets specifications.

It is important to consider hold-up volumes of not only the devices utilized in the process, but also of the system itself and the impact this has on overall process recoveries. In some cases systems are installed in cramped spaces, which may require device selection and physical attributes of the tubing/piping to include turns and differential in height. Along with hold-up volumes and system/piping design all of which contribute to frictional losses, fluid dynamics (viscosity, temperature, flowrate is another factor to help determine the system component requirements for each unit operation [5]. In addition, when scaling up, researching the type of hardware to be used at large scale is evident, however, at times, not given enough attention. For example, there are many devices out on the market that are fully encapsulated at small scale, but for equivalent larger scales these may require holders. Considerations of the large scale hardware systems must be addressed within the different unit operations. Some of these include physical attributes, automation and footprint. Finally, yet important, proper validation of the systems and process should be completed prior to scaling up or even manufacturing of clinical material.

In addition to specific facility and system needs, all of the unit operations share a common ground of considerations for implementation and tech transfers. One of these considerations is for companies to further investigate each molecule’s process operating conditions via Design of Experiments (DoE) or even a deeper dive into a Quality by Design (QbD) approach. Another consideration is to understand raw material and consumable lot-to-lot variability, and the processes batch to batch variability. These factors can provide a better understanding of each unit operation and the performance of the process as a whole, which can contribute to the robustness and possible higher degree/window of operation. In cases where these deeper approaches may not be feasible, an upfront investment of rationally defined safety factors can be incorporated for all unit operations to minimize the risk for process deviations [6].


5.0 Conclusions
Antibody variants such as Fabs, BsAb and fusion proteins are generating increased interest as the demand for target therapeutics with improved efficacy continues to grow. Compared to traditional MAB processes, these molecules present some developing and manufacturing challenges. Each of the steps in recovery and purification of these molecules must be optimized based on the process requirements and the molecule characteristics, ensuring robust, stable and scalable production processes.


January 9, 2015 | Claire Scanlan | Mireille Deschamps | Juan Castano | Ruta Waghmare, PhD | Corresponding Author Ruta Waghmare , PhD | ruta.waghmare@emdmillipore.com | doi: 10.14229/jadc.2015.1.9.001

Received: December 10, 2014 | Accepted January 7, 2014 | Published online January 9, 2014

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This work is published by InPress Media Group, LLC (Downstream Processing Considerations for Antibody Variant Therapeutics by Claire Scanlan, Mireille Deschamps, Juan Castano, Ruta Waghmare, PhD) 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.

Last Editorial Review: January 9, 2015

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Sorrento Therapeutics Completes Acquisition of Concortis Biosystems

Sorrento Therapeutics, Inc. announced today that it has completed its acquisition of Concortis Biosystems, Corp. The transaction provides Sorrento with a comprehensive antibody-drug conjugate technology platform designed to create a new generation of homogenous antibody drug conjugates (ADCs) with site-specific toxin conjugation and consistent drug-antibody ratios. With the close of the deal, Sorrento has issued an aggregate of 1,331,978 shares of its common stock to the Concortis shareholders. Based on the closing share price on December 18, 2013, the transaction is valued at $11.3 million.

With the acquisition, Sorrento adds Concortis’ proprietary cytotoxic payloads as well as C-lock® and K-lock® conjugation technologies that allow for site-specific toxin conjugation to the antibody. These new technologies may improve the overall stability and potency of the ADC. First-generation conjugation technologies lead to inconsistent drug-antibody ratios, which result in a heterogeneous mixture of ADCs. This variability has been a constraining factor in unlocking the full therapeutic potential for current-generation ADCs.

The ADC technology complements Sorrento’s existing development programs, particularly its G-MAB® antibody library. The G-MAB library contains a vast and diverse array of fully functional monoclonal antibodies, including antibodies that hit the historically difficult to target G-protein-coupled receptors (GPCRs). In 2015, Sorrento expects to begin clinical testing of its monoclonal antibody targeting PD-L1 as well as its ADC targeting VEGFR2.

The company has also developed a diverse library of fully functional monoclonal antibodies, complemented by a comprehensive ADC platform comprised of wholly owned conjugation chemistry, linkers and toxic payloads. Sorrento’s multi-pronged approach to combating cancer with small molecules, antibodies, and ADCs provides optionality in addressing the complexity of cancer.

“While our near term clinical opportunity exists in a bioequivalence trial of Cynviloq™ beginning in 2014, this ADC technology acquired from Concortis will help maximize the potential of our G-MAB library and fuel our R&D pipeline over the long-term,” said Henry Ji, Ph.D., President and CEO of Sorrento. “The successes of first-generation ADCs have paved a path for new conjugation technologies. Our ownership of each of the key components of an ADC – the antibody, conjugation chemistry, linkers, and toxins – gives us a distinct and unique advantage in the field.”

Other agreements
Earlier this year Sorrento also signed a non-exclusive research license agreement with Lonza for access to Lonza’s GS Xceed™ Gene Expression System. Sorrento will use the GS Xceed™ System in the generation of stable expression cell lines for the production and development of therapeutic antibodies in the company’s product portfolio.

This research license give Sorrento access to Lonza’s GS technology for use in Sorrento’s maturing pipeline of therapeutic antibodies in oncology, inflammation, and infectious disease. Sorrento has shown yields of up to 3g/L with the GS Xceed™ System, in the production of antibodies targeting the vascular epidermal growth factor receptor 2 (VEGFR2), an important anti-angiogenesis target for a number of solid tumor indications. Several monoclonal antibodies (mAbs) will be produced and developed under the new agreement, including Sorrento’s lead anti-PD-L1 and anti-PD-1 antibodies, as well as a selection of traditionally difficult-to-obtain anti-chemokine receptor mAbs.

“We license Lonza’s GS Xceed™ System for the development of our lead human therapeutic antibody programs,” commented Henry Ji, Ph.D., President and Chief Executive Officer of Sorrento Therapeutics. “The speed and ease of use of the GS Xceed™ System will aid in the rapid selection of high-producing cell lines and accelerate the production and development of our most promising therapeutic candidates.”

“We look forward to supporting Sorrento’s efforts to expand and develop its pipeline of promising new therapeutic candidates,” said Karen Fallen, VP of Licensing and Technology, Lonza Development Services. “The GS Xceed System™ is an innovative and robust expression system derived from our industry-leading original GS System™ optimized through years of process development expertise.”

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Investigational Antibody-Drug Conjugate, Shows Encouraging Results in Women With Highly Advanced HER2-positive Breast Cancer

Results of a Phase II study of trastuzumab (Herceptin®, Genentech) in combination with DM1 (T-DM1), an investigational HER2 antibody-drug conjugate being developed by Genentech, in collaboration with Roche and Immunogen, Inc., shows encouraging results in women with highly advanced HER2-positive breast cancer.

Positive Phase I and Phase II findings were first reported at the 2007 [1] and 2008 [2], respectively, annual meetings of the American Society of Clinical Oncology (ASCO) with T-DM1 in patients with HER2-positive (HER2+) metastatic breast cancer (MBC) that progressed on treatment with trastuzumab. In the Phase II study, 60% of patients also had been treated with lapatinib (Tykerb®, GlaxoSmithKline).

As assessed by independent review, the T-DM1 combination shrank the tumors (also known as objective response) in 33% of women with advanced (metastatic) HER2-positive breast cancer that had worsened following previous treatment. Women in the study had already received an average of seven drugs for metastatic disease, including chemotherapy, trastuzumab and lapatinib, prior to receiving T-DM1. No new or unexpected safety signals were observed. The results were presented today at the 32nd Annual CTRC-AACR San Antonio Breast Cancer Symposium (SABCS) from 9 to 13 December 2009 (Abstract #710). [3]

Antibody-drug conjugates or ADCs [4] are a unique combination of a precise and targeted monoclonal antibody, a stable linker, and a potent cytotoxic. They have broad utility in basic, preclinical, and clinical applications. [5] T-DM1 combines two approaches in one medicine: the anti-cancer activity of the trastuzumab antibody, which blocks signals that make the cancer more aggressive and signals the body’s immune system to destroy the cancerous cells, and the targeted delivery of the potent cytotoxic DM1.

“Breast cancer is the most common cancer among women worldwide with more than one million new cases diagnosed every year and nearly 400,000 deaths, so it is vital that we continue to provide more treatment options” said William M. Burns, CEO of Roche Pharma.

According to the American Cancer Society [6], breast cancer is the second leading cause of cancer death in the United States. Women diagnosed with advanced (metastatic) disease have a poor prognosis and only 27% survive five years.

Approximately 15 to 30% of breast cancers are HER2-positive. [7] When HER2-positive breast cancer is advanced, the disease has spread to other parts of the body, most commonly to the lungs, bones, liver and brain. At this stage of the disease, the current goals of existing treatments include symptom relief, tumor shrinkage, improved quality of life and increasing the amount of time women with advanced breast cancer live without the cancer worsening. There are no treatment guidelines or FDA-approved treatment options for women with advanced HER2-positive breast cancer if the disease progresses following treatment with trastuzumab and lapatinib.

“The much anticipated data on the investigational drug T-DM1 will be welcomed by physicians treating women with early and very advanced stages of breast cancer as it will offer them more choices for fighting this devastating disease,” Burns noted.

“Despite major advances in HER2-positive breast cancer, the disease may still progress after multiple treatments, to the point where there are no approved HER2-targeted medicines,” said Hal Barron, M.D., executive vice president, Global Development and chief medical officer, Genentech. “Results from this study are promising for women who need new treatment options, and we will discuss next steps of the T-DM1 development program with the FDA.”

“These results are significant because they demonstrate that T-DM1 was effective at shrinking tumors in women whose cancer had progressed following prior treatment with standard therapies for HER2-positive breast cancer,” said Ian Krop, M.D., Ph.D., a medical oncologist at Dana-Farber Cancer Institute, and lead investigator on the study. In this single-arm study, 45% of women experienced a clinical benefit (defined as a complete or partial tumor response, or stable disease, maintained for at least six months), as assessed by independent review. Adverse events were similar to those observed in previous clinical trials of T-DM1. The most common severe adverse events included thrombocytopenia (a low level of platelets in the blood, 5.5%) and back pain (3.6%), and the most common adverse events were fatigue (59.1%) and nausea (37.3%). No severe (Grade 3 or higher) cardiac-specific side effects were observed. One patient with pre-existing, non-alcoholic fatty liver disease died with hepatic failure.

Ongoing clinical trials
The new treatment options is currently in trial in a Phase II study, known as TDM4374g, a single-arm, multi-center trial designed to assess T-DM1 as a single agent in 110 women with HER2-positive advanced breast cancer whose disease had progressed after receiving at least two prior HER2-targeted treatments (trastuzumab and lapatinib) in the metastatic setting, as well as an anthracycline, a taxane, and capecitabine. [8]

The primary endpoint of this study is objective response rate (a complete or partial tumor shrinkage of at least 30%, determined by two tumor assessments at least 28 days apart), as measured by an independent review facility. Secondary endpoints include safety, clinical benefit rate, duration of response and progression-free survival (PFS).
Duration of response and PFS data are not yet mature and will be presented at a future meeting.

The results of the data presented during the SABCS follow on from results from another phase II study (TDM4258) presented at ASCO 2009 which also showed encouraging results in women with advanced HER2-positive breast cancer [9, 10].

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