Are some cell counts too good to be true? Why some companies' product data may mislead.

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This is a cautionary tale about the need for robust product characterization and release specifications for all cell therapy products.

Background

While our food often has a list of ingredients, our drugs don't.  We rely on our regulatory agencies to rule on the safety of our drugs.  These agencies require drug manufacturers to submit to them the composition of their therapeutic compounds and then to comply with the product specifications.  It is this composition and these specifications which formed the basis of the clinical data evaluated by the agency and upon which the marketing approval is based.  Any deviation from those specifications requires a submission to the regulatory agency for review. Any deviation without such a submission is punishable.   

At the manufacturing site, as products come off the line they are subjected to a panel of product release tests to ensure each batch complies with the product specifications.

Specification compliance is a direct function of the consistency of the raw and ancillary materials, equipment, and operating procedures used in the manufacturing process.

Cell therapies present unique challenges when complying with this paradigm for several reasons only two of which I will mention here.  Firstly, it is not possible to achieve the level of product purification as one might with other therapeutic products.  Secondly, the product characterization is at a cellular rather than molecular level.

Autologous cell therapies present another set of unique challenge in this paradigm because of the notable patient-to-patient variability where the patient is also the donor of the raw material.  This often means there is a wider tolerance of heterogeneity in the product but it still must be within what has been proven to the regulatory agency as a safe and effective range.  

In cases where an autologous cell therapy is centrally manufactured, they are most often subjected to product release testing similar to that described above.  One notable difference, particularly for fresh products, is that the products may be shipped to the clinic and even administered before the full panel of test results are obtained.  This wold be considered highly unusual (if ever acceptable) with other types of products but is tolerated because of the time-sensitivity of these products and their high safety profile.

In the case of autologous cell therapy products produced at the bedside there is often not the same kind of product release discipline.  Often the regulatory agencies deal with the product consistency and specification compliance issue by ensuring that the cell processing device used point-of-care is validated to ensure the cellular product output is always within a specified range shown to be clinically safe and effective.

The Varying Degree of Product Characterization/Specification of Autologous GTP Cell Therapy Products

However - and now I get to the point of this blog post - for cell-based products, procedures and/or devices/kits which are not mandated to be formally approved by a regulatory agency before they can be commercially marketed, there is no product specification rigor.  Compliance with the Good Tissue Practice regulations and guidance is deemed to ensure safety.  In the United States, cell-based products which are deemed to be "minimally manipulated" and intended for "homologous use" are typically allowed to go straight to market with no formal approval.  Safety and clinical data is not required but is practically necessary to support physician adoption and, where applicable, reimbursement.  

This means that for these products there is a great deal of variability in terms of how much rigor companies apply in characterizing their product and then ensuring that each batch complies with the specifications they themselves have determined to be safe and effective. Again, where such products are manufactured in a centralized facility the likelihood of some release testing is greater.  However, those companies relying on a point-of-care processing kit or device business model that has not been deemed to require formal market approval, rarely (if ever) include product release testing.

The common criticism of these companies is that they simply do not know what they are injecting into patients because of the combination of the patient-to-patient donor variability, the lack of any disciplined product characterization or dosing studies, and the absence of any product release testing.  

This criticism is not equally levied at all autologous GTP products or companies - even those relying on point-of-care processing.  Of course some companies care and do a lot to try to ensure their product is well-characterized and that each batch complies with product specifications. This may involve the use of product release tests but can also involve the combination of pre-market research into the product characterization, safety, and dosing along with validation of the device/kit output.  In this way a company can say that within a very small margin, the output will be within the product specifications the company knows is safe and efficacious.

However, in a rush to get their device/kit to market some companies appear to care very little about the cell product characterization, validation of the output of their device/kit, or tying this data to optimal dose.

More concerning are those companies that appear to provide such data but it is wrong or meaningless.  What follows appears to potentially be a case study of precisely this problem. 

The INCELL Study 

This week I came across a fascinating white paper from Incell Corporation analyzing the output of adipose tissue processing kits of MediVet-America apparently demonstrating the inaccuracy of their cell counts (a common type of cell therapy product characterization) and calling into the question the cell count claims of Intellicell Biosciences (New York, NY) and Adistem (Hong Kong).

At the heart of the critique is the claim that the cell counting (product characterization) techniques employed by these companies counts as cells things (namely acellular micelles) which are not cells.

I encourage you to read the white paper in its entirety.  They corresponding author told me to watch for one or more papers which they are preparing for submission to peer-reviewed publications shortly.  Presumably these will rely on a larger data set and perhaps test other methodologies or technologies.

For the purposes of this blog, I've pulled what I believe are the most salient excerpts below:

Intrigued by the high cell numbers  (5 to 20 million cells/gram)  reported by kit/device manufacturers such as MediVet-America (Lexington, KY), Intellicell  Biosciences (New York, NY), and Adistem, Ltd. (Hong Kong) in adipose stem cell therapy compared to other methods (e.g.,  Chung,Vidal, and Yoshimura), INCELL staff conducted a research study to  investigate the high apparent yield of stem cells.  This initial work was focused  on SVF cells from the MediVet Kit, which is marketed to isolate adiposederived canine SVF and stem cells.

The cell yields reported for the Medivet Kits are five to more than ten times higher than the yields routinely obtained by INCELL from freshly harvested human or animal adipose tissue using our adipose tissue processing methods.  These yields are also tenfold or higher than those reported in the literature by most academic researchers (Chung-canine, Vidal–equine, Yoshimura–human).  Since these  cell counts are used to support stem cell dosing recommendations and cell banking, it is important to better understand why the cell numbers are higher.

...

A comparative analytical study of three dog donors of adipose tissue was designed to evaluate the cell yields using the MediVet Kit as an example of this class of isolation system. All  kit procedures were followed as per the instructions provided.  A brief overview of the different cell counting methods used, and the resultant cell counts, observations and explanations of the results observed, are described below

....

This study shows that incorrect counting of adipose derived SVF cells and the subset of regenerative stem cells can subsequently result in inaccurate dosing, both in direct therapeutic applications and in cryostorage of cells for future use.  The DAPI-hemocytometer cell count (manual) was considered the most accurate, but there are various sources of technical difficulties that  can lead to incorrect  cell numbers. The nature of adipose tissue itself with variability in dissociation by enzymatic digestion can all contribute to the outcomes. Fat tissue has a propensity to form acellular micelles and oils upon tissue disruption. Processing methods or reagents (e.g., Solution E or lecithins) can generate micelles that may be  erroneously  counted as cells. Autofluorescence and dye trapping or uptake by the micelles can lead to very high inaccurate cell counts when automated cell counting is used. 

In this study the most inaccurate counting came from the Cellometer. When used according to kitrecommended guidelines and on-site training provided by Nexelcom for counting  cells by the MediVet procedure, the Cellometer overstated the DAPI-hemocytometer cell count by up to 20X or more. The Coulter Counter protocols also led to incorrect, high cell numbers. Although the cell counts were still a bit high, the authors recommend the NucleoCounter, or similar equipment, as more acceptable for automated counting.  The manual hemocytometer-DAPI method is the most accurate, but requires a highly experienced cell biologist or technician to make accurate counts and  is not suitable for routine clinical use. 

...

Other companies also have claims of very high cell numbers when their processes are used. Adistem, like MediVet, states they add an emulsifying agent to their kits to assist in cell release, and they also use a light activation system. Their kits were not tested in this study but it is possible that the high cell numbers reported by Adistem are also incorrect and result from the same problems highlighted in this paper for the MediVet procedure. Ultrasonic energy, which is commonly used to manufacture micellular  liposome  structures and to disrupt and lyse cells, is  another potentially problematic procedure for counting and verifying viable, regenerative cells.  Intellicell 3uses ultrasonic energy to release cells from adipose tissue, and it is possible that resultant micelles or cell fragments contribute to the higher than expected cell numbers.  This assumption could be verified with additional studies.  

In summary, the authors caution that great care must be taken when using kits and automated cell counting for stem cell dosing and cryobanking of cells intended for clinical use. Overestimated  cell numbers would be a major confounding source of variation when efficacy of stem cells injected are compared as doses based on cell number and when cryostored cells are aliquoted for use based on 

specific cell numbers as a treatment dose.  Hopefully, this study will lead to more  reproducible counting and processing methods being reported in the literature, more inter-study comparability of cell doses to clinical outcomes,  more industry diligence to support claims, and more accurate counting for dosing stem cell therapies to patients.

...

Chung D, Hayashi K, Toupadakis A, et al.  Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia.  Res Vet Sci, 2010; 92(1):66-75. Vidal MA, Kilroy GE, Lopez MJ, Johnson JR, Moore RM, Gimble JM. Characterization of equine adipose tissue-derived stromal cells: adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells. Vet Surg, 2007; 36:613–622.  Yoshimura K, Shigeura T, Matsumoto D, et al:  Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirate.  J Cell Phys, 2006; 205:64-76.

 In Conclusion

Despite some of their other challenges, Intellicell, MediVet-America, and AdiStem (and others) have scored credibility points with some of my colleagues who have been impressed by the fact that they have incorporated product release criterion and testing technologies into their business model where their peer companies have not bothered.  This credibility may be quickly eroded if it turns out the results of their cell counts have been misleading.  For now it is a word of caution to do your own due diligence and/or not to fall into a similar product development/characterization trap.  Meanwhile, we will watch for the peer-reviewed papers.

Potential far-reaching implications of the ongoing fight over point-of-care autologous cell therapy

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The FDA recently issued an untitled letter to Parcell Laboratories and its contract manufacturing organization, New England Cryogenic Center (NECC), pertaining to the product PureGen™ Osteoprogenitor Cell Allograft intended for the "repair, replacement, reconstruction of musculoskeletal defects". The letter stated:

"The PureGen™ Osteoprogenitor Cell Allograft is not the subject of an approved biologics license application (BLA) nor is there an IND in effect. Based on this information, we have determined that your actions have violated the Act and the PHS Act."

This would appear to be an indicator that the FDA does not intend to relax enforcement of its view of how autologous cell therapies are to be regulated despite its ongoing litigation on this very subject with RSI.

As followers of this blog know, since the battle's inception in 2008 I have followed the case of Regenerative Sciences, Inc and their war with the FDA over their right to provide certain autologous cell therapy treatments to patients in certain circumstances without FDA approval. My first blog entry on the topic was in September 2008 in which I pointed out that the FDA had written a letter to RSI that July taking issue with some of their practices.

My next blog on the subject was February 2009 in which I concluded "I think the FDA is building its case and a showdown is on its way to Denver-town."

At the advice of legal counsel, I pointed out in a March 2010 blog entry that my reference to FDA's July 2008 letter to RSI was not officially a "warning letter" as that is defined and as I had referred to it but rather an "untitled letter". I also commented that FDA's lack of enforcement action against RSI to-date was emboldening medical practitioners into thinking FDA was reconsidering their position on the legality of providing autologous, expanded cells to patients outside of an FDA-cleared IND or BLA.

I took some satisfaction in announcing on my blog in August 2010 that the FDA had finally taken action against RSI. My satisfaction was not rooted in a belief that the FDA is right (I've always been agnostic as to which side is right) but in the sense that things had occurred as I had predicted they would.

The action the FDA chose to take against RSI was to seek an injunction against RSI from continuing to provide the "offending" treatment - a version of the Regenexx™ procedure using mesenchymal stem cells (“MSCs”) grown outside the body after harvest for the later infusion back into the donor-patient for the treatment of various orthopedic conditions - which the FDA alleges is a "product" falling under its regulatory authority but for which RSI has never received any FDA clearance to provide to patients.

RSI counterclaimed against the United States, challenging the FDA’s authority to regulate the Regenexx™ Procedure in question and challenging certain FDA regulations. The United States moved to dismiss Defendants’counterclaims and for summary judgment.

I have continued to follow the case relatively closely through a number of source (see this sample media coverage in OthosSpineNews) as it continued to progress. Last month, for example, a blog I follow posted an eloquent case in support of RSI's position with the help of Mary Ann Chirba, J.D., D.Sc., M.P.H. of Boston College Law School.

So it was with much interest that I was recently notified of an order issued by the court which appears to have the potential to take the case in a very unexpected direction with enormous potential ramifications.

The context is that the judge was reviewing the FDA's motion for summary judgment, RSI's response, and FDA's reply when the judge issued this order to show cause.

At its essence the Judge has ordered the FDA to file a brief no later than 26 September showing the court why the term “chemical action” applies to stem cells. It is is a short Order the core of which reads as follows:

The Government finds its definition for a “drug” in the FDCA: “The term ‘drug’means . . . articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals” and “articles (other than food) intended to affect the structure or any function of the body of man or other animals.” 21 U.S.C. § 321(g)(1)(B)&(C).

This definition, at least as to subsection (C), would be broad enough to encompass a boot on a patient’s ankle to hold it secure after ankle surgery. The Court doubts that was Congress’s intent.

Neither party references the definition for “device,” found in the statute at 21 U.S.C. § 321(h). A “device,” is a certain kind of “article” used in diagnosis, cure, mitigation, treatment or prevention of disease, 21 U.S.C. § 321(h)(2), but which, presumably unlike a drug, “does not achieve its primary intended purposes through chemical action within or on the body of man or other animals and which is not dependent upon being metabolized for the achievement of its primary intended purposes.” 21 U.S.C. § 321(h).

These contrasting definitions immediately raise the question of why the Court should not interpret the meaning of the word “drug” to include not only an article for use in diagnosis, etc., and intended to affect the structure or function of a patient, but also an article that “achieve[s] its primary intended purposes through chemical action” and which is “dependent upon being metabolized for the achievement of its primary intended purposes.” Id.

The United States is ORDERED TO SHOW CAUSE why the Court should not read the definition of “device” at 21 U.S.C. § 321(h) as informing and restricting the definition of “drug” at 21 U.S.C. § 321(g)(1)(B)&(C).

It will be most curious to see how the FDA argues out of the corner that many believe the Judge has painted the agency. The FDA recently defined “chemical action” in its draft "Guidance for Industry and FDA Staff: Interpretation of the Term “Chemical Action” in the Definition of Device under Section 201(h)of the Federal Food, Drug, and Cosmetic Act". What is curiously absent from the document is any mention of cells or HCT/P’s despite CBER’s approval stamp on the document.

Another line of argument centers around whether cells - notably 'stem' cells - are “metabolized" as that term is defind.

If one extrapolates the ramifications of where the court appears to be currently leaning, the implications of this judgment may have far-reaching implications for biologics in general well beyond cell therapy and certainly well beyond autologous cell therapy.

As some quite logically argue, one potential scenario is that this judge rules all biologics fail to fall within the legislative "drug" definition. The argument goes like this. The drug regulations live under title 21, which has narrow definitions for what constitutes a drug. The FDA’s authority over biologics comes from title 42, which is merely to control communicable disease transmission in transplants, with no authority to take the drug provisions from title 21 and apply them to title 42. So the agency is risking a loss of control over all biologics.

What’s curious here - and perhaps somewhat ironic for RSI at this stage - is that they only ever set out to challenge their ability to regulate autologous cells used by a physician as part of his or her medical practice yet now the FDA's authority to govern all biologics is currently under question.

The fact that I cannot fathom the courts striking FDA's jurisdiction over all biologics when the dust settles on this case does not make the arguments any less compelling and it does leave open the possibility that a lower court Judge such as the one presiding over this case may be inclined to make a ruling which essentially ensures the issues are punted to the appellate courts for a more considered ruling. In such circumstances, even if the FDA were to prevail at the end of the day (perhaps a decade down road) the uncertainty such a ruling would rain down on the sector would be commercially stifling - even if if were just limited to autologous cell therapy let alone if were any broader.

My dated and unpolished law degree can only take this analysis so far and anyone interested in some further but delightfully light and practical reading on the potential ramifications of the case could do no better than read a paper published recently by the relevant practice groups at the law firm K&L Gates entitled "Cultured Stem Cells for Autologous Use:Practice of Medicine or FDA Regulated Drug and Biological Product in which they review the case and its potential implications - the latter of which the authors are not guilty of underestimating in the following concluding sentence of their analysis:

The court’s decision will, to a large degree, dictate the types of legal strategies and business models that will be necessary to successfully perform stem cell procedures in the future.

_____________

post-script: The potential stink of commercial uncertainty wafting from this case is even more egregious when combined with the uncertainty around what to expect from the FDA in its much-anticipated and typically overdue guidance on adipose-derived cell therapies.

Rumor has it that the FDA is leaning toward considering most (if not all) means of deriving cell populations from adipose tissue (typically lipoaspirate) to be governed as what we colloquially refer to as a '351' thus taking it out of the purview of the practicing physician and into the hands of companies prepared to follow the traditional "drug development' model for new medicines. The rationale here is that the mechanical and/or enzymatic digestion required to separate the desired cell populations from the stroma take the process beyond "minimal manipulation'.

Watch for this guidance from CBER OCTGT in the weeks to come and/or any relevant rulings by the Tissue Reference Group. This would be a serious blow to those building business models around point-of-care, autologous adipose-derived cell therapy treatments.

What makes this even more interesting is the pace of which US-based medical practitioners (and/or companies supporting them) are adopting and selling autologous cell-based products, services and/or treatments for sundry indications in ways which many would argue are apparently in obvious and flagrant disregard for the FDA's regulatory authority over such treatments. Included for consideration on such a list would be the following:

IntelliCell Biosceinces

eternaMD

Arizona Stem Cell Center

.