Challenges in the Characterization of Antibody Drug Conjugates

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories
www.abclabs.com

Antibody Drug Conjugates (ADC) provide a unique new treatment for a variety of cancers. ADCs consist of a monoclonal antibody (mAb) targeted to the receptor of interest and linked to a highly cytotoxic payload. The mAb binds to the cell and enters the cytoplasm. Once inside the cell, the linker is cleaved and the toxin released. This provides the ability to use highly cytotoxic compounds without the serious side effects of systematic chemotherapy. It is estimated that there are 100-150 ADCs currently in preclinical development. ADCs present unique analytical challenges.

In addition to the complicated mAb, there is the added complexity of combining a cleavable linker and a cytotoxic drug. This introduces the necessity for determining drug loading, linkage sites and Drug Antibody Ratio (DAR). The critical technique for analysis on ADCs is ultra-high resolution QToF mass spectroscopy. This allows for determination of relative loading, DAR and linkage sites, as well as PTMs, disulfide linkages and related substances/degradants (deamidation/oxidation, truncations, and amino acid substitutions). Due to their high resolution, UHR-QTof instruments have the ability to sequence proteins up to 25-30 kDa. The specific protease IdeS (which cleaves just below the hinge region of IgG) under reducing conditions results in three polypeptide chains of ~ 25kDa: Fd region, Fc/2 region and the LC region. Analysis of these digests by UHR QTof MS yields complete sequencing of each polypeptide, DAR, payload and glycan distribution. If necessary, other proteases may be utilized for more detailed analysis. Additional characterization includes the following:

• Intact mass
• Deglycosylated mass
• IEX
• Imaging CIEF
• CE-SDS
• SEC
• N-linked glycan analysis
• HIC (secondary DAR analysis)
• Binding assay
• Bioassay
• Higher Order Analysis (CD, AUC, DSC, etc.)

The FDA considers the mAb a drug substance so both the mAb and ADC must be fully characterized. In addition, the agency has been requesting characterization of charge variants of the mAb and ADC. This necessitates preparative IEX followed by characterization of the acidic and basic fractions. Based on all these considerations, characterization of ADCs require careful planning and attention to detail.

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Analytical Method Development: Beware the Rabbit Hole

By Wayland Rushing, Ph.D.

Senior Scientific Advisor

ABC Laboratories

www.abclabs.com

Developing analytical methods for pharmaceutical analysis can be one of the most time consuming and rewarding experiences in today’s analytical lab. I fondly recall the days (from many years ago) of sitting in front of the HPLC anxiously awaiting the data from the latest run for evaluation.  Either hours/days of hard work were about to be vindicated or hopes dashed and it was back to the drawing boards.  This scenario is still occurs on a daily basis throughout our industry. Unfortunately there is a potential dark side to the developmental process.  It can be very easy for the method developer to get lost in the task, ever trying to develop the “perfect” analytical method.  I have watched many analytical chemists get caught in this web of ever trying to catch this unicorn.  There is never a perfect method, it can always be improved, accuracy tweaked, precision optimized, etc.  So how do we know when enough is enough?

Let’s take the development of an impurities method as an example. The impurity is present at 0.15% and has a specification of 0.5%.  We are going to charge our developer with developing a method to quantitate for the impurity and we turn him lose.  After a couple of weeks we touch base with them and are updated the method isn’t ready, so development continues, couple more weeks pass, then another month, then finally a method is delivered.  The method can see the impurity down to 0.005%, has recovery of >99% with <1% RSD at the quantitation level.  And we stand amazed, not only at the performance of the method (quite impressive), but also finally realizing the time and money spent on a developing a method that far exceeds what it needed to do.  Simply put, for an impurity method, this is overkill, this level of method performance simply isn’t needed.

I have watched this play out in the development arena multiple times over again.  With the main drivers of development being time, quality and cost we must learn how to balance all of them to achieve the goals of development.  The mistake made here is that the development of the method wasn’t guided, there were no expectations set on what the intent of the method should be and what level of performance the method should have.  When initiating any analytical development there should be key questions that are asked and answered before any labwork commences:

• What is the intent of the method?
•  How low does the method need to go down to?
• What level of accuracy is needed?
• How precise does the method need to be?
• What is the range of the method?

So lets re-evaluate our development plan for the above example and answer the above questions first:

• What is the intent of the method?
• Answer: to quantitate the impurity in question for release and stability testing.
• How low does the method need to go down to?
• Answer: the method needs to have an Limit of Quantitation of 0.05%
• What level of accuracy is needed?
• Answer: At the specification the method should have an accuracy between 90 – 110%.
• How precise does the method need to be?
• Answer: At the specification the method should have no greater than 10% RSD.
• What is the range of the method?
• Answer: the method should be linear from 0.05% to 0.75%.

If we apply these questions to the above development we not that the method developer had met these conditions within the first two weeks of the development program.  But his level of understanding of what the method needed to do and the actual needs of the method were not aligned and hence spending an additional couple of months in development, wasting time and money.  This is a common occurrence which is easily preventable, set expectations before you start the work.  Ensure that your internal analyst or the CRO you are working with understands the difference between development a perfect method and developing a method which is suitable for its intended use.  This can often be the difference between having an efficient development program and one that suffers from unexpected delays in time and budget excesses.

21st Century Cures Act – Is It Truly Progress?

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories
www.abclabs.com

Dr. Francis Collins, Director of NIH, has stated that it takes “around 14 years and $2 billion or more “to develop a new drug. Based on statements like these, Congress has come to the conclusion that the drug approval process is too onerous and lengthy and is the primary impediment to the discovery and approval of new drugs. In response they have passed the 21^st Century Cures Act with nearly unanimous, bipartisan approval. But is this a fallacious assumption? Some argue that the FDA review and approval process is the fastest and most streamlined in the world and that accelerating the approval process may impact patient safety.

In an editorial in the New England Journal of Medicine, Dr. Jerry Arvon states ”The bill would also encourage the FDA to rely more on biomarkers and other surrogate measures rather than actual clinical end points in assessing the efficacy of both drugs and devices.” While surrogates have previously been utilized as endpoints by the FDA to support accelerated approvals, this was only for drugs intended to treat life threatening illnesses.  Dr. Arvon goes on to state that biomarkers “may not always predict the drug’s capacity to improve patient outcomes “. The literature has many examples of cancer drugs approved utilizing these criteria that not only do not extend life, but have side effects.

While the argument regarding expedited drug approval continues, one aspect of the new law has unanimous support; an additional $8.75 billion in funding for NIH over the next 5 years.  According to a study published in Health Affairs, federal funding of basic research played a major role in the origin of transformative drugs approved between 1984 and 2009.

Pay now or Later? Early Method Development and Phase-Appropriate Validation-Considerations and Issues for Pharmaceutical Drug Development

By Harley Everett Wilcox, MBA

Senior Scientific Advisor

ABC Laboratories
www.abclabs.com

I do not know an explicit percentage, but I would guess roughly 30% - 50% of early purity methods for drug products have issues prior to phase II clinical.  The issue often observed include:

• New chromatographic peaks showing up in the drug product during stability

• Peaks migrating, or moving around over time

• System Suitability issues over time

Often, minimal efforts are put into early HPLC methods as the drug product may change and extra efforts would appear to be not useful and down-right costly.  In addition, time-lines are often tight. Additional method development efforts suggested for methods supporting phase one studies should be in concert with the established specifications and thus the issue.  Drug product specifications are required for release and stability monitoring and typical determined as the IND nears. If little is known about a new formulation, degradation profiles, and purity of lab/engineering, batches how does one determine purity specifications? Often, ICH guidelines or drug substance data are referenced for total impurities of NMT 2% and no unknown impurity greater than 0.2% represent typical specifications. OK, now we have specifications and we validate the early development methods as phase appropriate conducting minimal accuracy, linearity, precisions, stability of solution, LOD and LOQ.  We stay away from robustness, intermediate precisions, and complete specificity which would could be completed probably in a few weeks. Why do phase appropriate? Again, as things may change the cost would be sunk.

Often and unfortunately, either at release or during stability the analyst will encounter an investigation as the product does not meet specification because of:

• New peaks > unknowns specification

• New peaks, or higher than expected impurities from early manufacturing

Drug developers will exclaim ‘those unknowns are very likely excipient or excipients related’ and thus not a real OOS, or ‘I thought the method was validated’.  However, as the unknown peaks move around from run to run, and often have insufficient resolution from known active impurities, more work is necessary to confirm the unknowns as excipients or not. Typically, this requires evaluating placebo, placebo on stability, stressed excipients and possible some excipient drug compatibility studies. Gains made from the savings for early development/phase appropriateness are lost and unless more efforts are applied to understand the method, we may revisit costly investigations in the future.  The message—it is most cost effective to insure that methods and specifications are compatible and or conduct appropriate studies during method development to understand degradents and representative impurity levels.