Biopreservation Best Practices for Regenerative Medicine GMP Manufacturing & Focus on Optimized Biopreservation Media

CELL & GENE THERAPY INSIGHTS

LATEST ADVANCES IN ADDRESSING BIOPRESERVATION CHALLENGES

INNOVATOR INSIGHT Biopreservation Best Practices for regenerative medicine GMP manufacturing & focus on optimized biopreservation media

Brian J Hawkins, Alireza Abazari & Aby J Mathew

Cellular therapies are cell and tissue products sourced from biological materials that are employed as ‘living drugs.’ Such ‘living drugs’ require specialized biological support, namely biopreservation, to maintain op- timal recovery, viability and return to function post-preservation. To achieve successful biopreservation, optimization of a multitude of parameters, including cooling/thawing rates based on the biophysics of specific cell types, the temperature of storage and transportation, as well as biopreservation media are of paramount importance. In particular, the choice of biopreservation media is pivotal for clearing regulatory hurdles and facilitating commercialization. Traditional extracellular-like (isotonic), home-brew cocktails (which may contain serum) may not be compat- ible with a Good Manufacturing Practices (GMP) clinical manufacturing process. This article will address the challenges associated with main- taining the viable recovery and functionality of ‘living drugs’, discuss the benefits and consequences of low-temperature biopreservation, outline Biopreservation Best Practices, and propose considerations for incorpo- rating Biopreservation Best Practices into a GMP cell therapy product. Integration of Biopreservation Best Practices, beginning with the selection of optimized media, ensures that cells remain viable and functional throughout the cell product lifecycle, thereby optimizing manufacturing and clinical outcomes.

Submitted for Review: 10 Apr 2017 u Published: 5 Jun 2017

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Average global life expectancy has this spotlight issue. Rafiq and Coop- accelerated by 5 years at the fast- man describe considerations of bio- est growth rate since 1950 between preservation that emerge with man- 2000 and 2015, and now exceeds ufacturing scale-up. Stacey speaks 71 years [1]. However, with this in- to standards and quality attributes crease in lifespan comes a growing for cryopreservation of stem cells, incidence of chronic human diseas- which are more difficult to define es prevalent in aging, such as can- than in normothermic cell models cer, cardiovascular and neurodegen- and have proven variable and more erative diseases. As such, sustained complex within biopreservation progress in extending life expectan- cell models. Fuller provides cryo- cy and quality of life requires the biology principles to allow further continued development of novel understanding into the complex therapies and approaches that ad- biophysical considerations at low dress chronic human conditions temperatures. This collection of ex- prevalent in an aging population. pert perspectives regarding biopres- A promising development in the ervation, and related considerations treatment of chronic diseases is the to the scientific background, Good use of living cells as ‘smart drugs’ Manufacturing Practices (GMP), that can effectively target specific Quality/Regulatory attributes, and tissues to rescue organ function or translation to clinical applications, eradicate tumors. In the case of can- is a unique and valuable resource for cer, cell therapies have proven effec- clinical and commercial developers tive in patients who have exhausted of cellular therapies and regenera- other lines of standard care treat- tive medicine applications. ment, with reported results including complete remission and exten- sion of median survival time [2,3]. As living drugs, cell therapies need THE CHALLENGE to be viable and functional in order OF CELL THERAPY to be effective, and methods for pre- COMMERCIALIZATION serving small molecules and mono- Cell therapies originate from bio- clonal antibodies are not similarly logical sources, such as tissue biop- effective for cell-based products. sies, blood and bone marrow. This Rather, they require cellular support biological seed material is then ma- during all phases of the product life- nipulated in a laboratory or manu- cycle, from source material acquisi- facturing facility to develop the cell tion to final delivery and patient ad- therapy product. Whether autol- ministration. Optimized strategies ogous or allogeneic, the develop- to maintain the viable recovery and ment and eventual introduction of efficacy of cell therapies during all the therapeutic dose into a patient stages of collection, manufacturing involves rounds of transportation and delivery are essential to realize between the collection site and the the potential of these promising laboratory or cell manufacturing medical advances (Figure 1). facility, as well as storage periods The critical focus of biopreserva- of varying durations for logistic tion, or product stability, is reflected arrangements and quality control in the broad topics and expertise that checks. Unlike pharmaceutical or are reported as perspectives within small molecule agents, cells and

346 DOI: 10.18609/cgti.2017.035 innovator insight

ffFIGURE 1 Biopreservation practices.

Common traditional practice

Manufactured cell/tissue product

Collection Transport Processing Storage Transport Application

Source material Biopreservation

Preservation only considered for ﬁnished product Address yield issues AFTER negative system impact

Recommended Best Practices

Manufactured cell/tissue product

Collection Transport Processing Storage Transport Application

Source Material

Relevant biopreservation continuum

Biopreservation considered from source material to delivered product Do not wait until late clinical or commercial stages to optimize

Common traditional biopreservation methods focus only on the finished product. Biopreservation Best Practices recommends optimizing biopreservation and reducing stability stresses from collection of source material through final cell/tissue product application. tissues have unique requirements critical step in the product lifecycle. in order to remain viable and func- In many cases, the sites of collection tional while outside the body or and processing may be physical- culture conditions. Therefore, an es- ly conjoined or in close proximity, sential component of the cell man- ensuring cellular products can be ufacturing lifecycle is to preserve transported and manipulated with cells during handling and storage. little impact to stability. As preclin- More so, improper preservation of ical product development progress- biologic specimens at any step in es, the process typically requires the the process may negatively impact need for more operators and re- all subsequent manufacturing steps, sources, and may require additional and may adversely impact the final collection or processing sites. Cell cell therapy product. handling and transportation to re- During the early developmental mote sites therefore becomes more phases of cell therapies, the storage complex with increased risk poten- and handling of cells is often not a tial. In a large-scale clinical trial, the

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collection and processing of hun- tissues remain outside of normo- dreds of patients may be spread be- thermic conditions. For commer- tween remote sites. At this stage, an cial cell therapy products, effective optimized means to store and trans- biopreservation protocols provide port cell-based products between flexibility in manufacturing and remote collection and manufactur- shipping, facilitate effective process ing sites becomes more critical for development, and reduce manu- minimizing variability, increasing facturing costs. An ideal biopres- clinical efficacy and ensuring com- ervation protocol would maintain mercial viability. biological function throughout the product lifecycle, providing ‘vein- to-vein’/‘needle-to-needle’ support from the time the sample is collect- LOW-TEMPERATURE ed from the donor to the time it is BIOPRESERVATION administered to the recipient. CONSIDERATIONS: Under normothermic condi- HYPOTHERMIC & tions, cells must continually gen- CRYOGENIC CONDITIONS erate energy in the form of ATP As soon as biological specimens to maintain the intracellular envi- are removed from the body and ronment and permit essential en- normothermic conditions, dele- zymatic reactions. Indeed, approx- terious environmental stresses be- imately 20% of energy production gin to degrade the source material. is used to drive selective ion pumps From a clinical efficacy standpoint, that maintain the intracellular ion- these environmental stresses are ic balance and resting membrane compounded at each step of the potential [6], a figure that can rise cell product lifecycle, introducing to approximately 66% in metabol- sample variability and the potential ically active neural tissue [7]. Cells for impaired therapeutic function, specifically regulate the transport which may even lead to clinical of sodium (Na+), potassium (K+) inefficacy and termination of a -po and calcium (Ca2+) ions across the tentially effective treatment. From plasma membrane, store additional a financial standpoint, loss of cell Ca2+ ions within the endoplasmic yield, viability and function adds reticulum, and effectively sequester significant cost to cell therapies by K+ within the cytoplasmic compart- increasing the potential for repeat ment. The energy required to main- sampling or additional processing tain these ion gradients is generated steps to expand cell numbers or re- within the cell by mitochondria, suscitate function [4]. Biopreserva- which convert glucose and other tion refers to the processes required substrates into ATP through an ox- to maintain the health and function ygen dependent process. Reactive of biologics outside the body, as well oxygen species are a natural byprod- as suppress the degradation of these uct of energy production, but are ef- biological materials to ensure a re- fectively countered by cellular anti- turn to function post-preservation oxidant mechanisms of defense [8]. [5]. By reducing the stresses expe- Maintenance of cellular metabo- rienced by cells and tissues ex vivo, lism outside the body or dedicat- optimized biopreservation processes ed cell culture facilities under nor- can extend the time that cells and mothermic conditions would be

348 DOI: 10.18609/cgti.2017.035 innovator insight impractical, and would require a environment [12]. Under normo- continual oxygen supply, sufficient thermic conditions, this flow of nutrients to maintain metabolism, ions across the plasma membrane and a means to remove waste prod- would be controlled by ATP-de- ucts. In contrast to normothermia, pendent membrane pumps. How- each 10°C decrease in temperature ever, low temperatures also slow reduces metabolism approximately mitochondrial metabolism and fa- 50% for energetically active tissue cilitate the depletion of the cellular such as the brain [9], minimizing ATP levels necessary to fuel these the environmental requirements ATP-dependent membrane pumps. of cells outside culture conditions. In addition, impaired mitochondri- This Q10 coefficient reduction al function results in the increased in metabolism limits the need for generation of damaging oxygen free oxygen and substrates, minimizes radicals that may exceed the cell an- waste production, preserves cellu- tioxidant scavenging capacity [13]. lar ATP levels and attenuates the Although a small amount of ener- molecular processes that contrib- gy can be generated anaerobically ute to ischemic injury both during extra-mitochondrially by the break- storage and upon a return to nor- down of glucose via glycolysis, the mothermia post-preservation. As resultant formation of lactic acid a result, low-temperature biopres- lowers pH and triggers further cel- ervation, both at 2–8°C hypother- lular damage [11]. Unregulated ion mic temperatures, or cryogenically movement, combined with slowed frozen between -80°C and -196°C, membrane pumps, results in inter- is the most common and preferred ruption of the delicate intracellu- method of storage employed in cell lar ionic balance as well as osmotic therapy and regenerative medicine cell swelling. Further reduction in applications. temperature to below the freezing Despite the metabolic benefits point can induce the formation of of low-temperature biopreserva- intracellular ice crystals that can tion, temperature reduction exerts physically damage cells further by unique stresses that must be care- puncturing membranes and dis- fully addressed to maximize cell vi- rupting intracellular structures. ability and function upon a return More importantly, growth of intra- to normothermia. Cells at reduced cellular and extracellular ice crystals temperatures (hypothermic storage during freezing results in continued and cryopreservation) undergo re- concentration of salt ions and shift- duced membrane ion pump activi- ing pH and salinity, that adversely, ty and a physical reorganization of and sometimes irreversibly, impact the plasma membrane that increases intracellular and membrane pro- permeability [10]. Consequential- teins. Such cold-induced stresses, ly, Na+ ions flow into the cell and combined with a reduced ability to K+ ions escape into the extracel- scavenge free radicals, can accumu- lular compartment [11]. The Ca2+ late to levels that induce cell death. concentration inside the cell also While on the surface hypothermic rises over 1000-fold due to both a and frozen storage may appear to release of Ca2+ from the endoplas- impart divergent stresses, the tran- mic reticulum stores, and an in- sition to, through, and from the flux of Ca2+ from the extracellular frozen state exerts hypothermic

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stresses on cells. Indeed, cells can survive have likely undergone pop- experience profound supercooling ulation selection, potential genetic (i.e. non-frozen temperatures be- drift and may exhibit adaptations low the freezing point of a solution) that negatively influence down- of between 10 and 20°C, depend- stream cellular function in clinical ing on the freezing rate, before the applications. Ultimately, after low advent of ice formation [14]. Fur- temperature biopreservation, the thermore, the non-frozen fraction ordered priorities of the cell are sur- during cryopreservation experienc- vival, repair and recovery, and then es a hypothermic continuum until functional return. This is relevant reaching the vitrified state below in the context of cell therapy prod- the glass transition temperature. ucts, which are expected to func- As such, hypothermic stresses are tion upon thaw and delivery to the ever-present throughout the hypo- patient. thermic continuum regardless of whether the sample is in the frozen or non-frozen state, and can con- tribute to the damage observed fol- OPTIMIZED lowing cryopreservation [15]. BIOPRESERVATION Even cells that do not lyse during METHODS IMPROVE hypothermic/cryogenic tempera- CELL VIABILITY & ture exposure are sometimes irre- FUNCTIONALITY versibly damaged after a return to While the biopreservation of cell normothermic temperatures. A therapy products appears daunting, certain percentage of cells are dam- many of the stresses imposed by low aged to the point that they will temperature biopreservation can perish over time – by necrosis, pro- be mitigated by optimizing meth- grammed apoptosis and secondary ods and storage media. In practice, necrotic cell death – in a process there are numerous stress points known as Delayed Onset Cell Death that occur during the workflow(Fig - [16,17]. Indeed, Stroncek et al. re- ure 1), and transition to and from ported that transduced peripheral low temperature biopreservation at blood lymphocytes apparently via- both hypothermic and frozen con- ble post-preservation exhibit a de- ditions. For hypothermic biopres- cline in survival over several days ervation, critical steps include the in culture, although at the time the choice of storage media at relevant potential link to Delayed Onset stages, the rate and temperature of Cell Death was not established [18]. solution addition, the storage tem- While cell loss can be mitigated by perature, the warming rate and, additional culture in the laboratory ultimately, the removal or dilution or cell manufacturing facility [19], of the storage solution. As opposed expanded post-preservation culture to hypothermic storage, cryopres- would necessitate remote cell cul- ervation imparts additional stress ture facilities at the clinic and a de- points related to ice formation and lay in patient administration, both includes, in sequential order, the se- of which add significant financial lection of an appropriate biopreser- costs and could render the cell ther- vation solution and cryoprotectant, apy non-viable from a commercial the rate of cryoprotectant addition, standpoint. In addition, cells that the temperature of cryoprotectant

350 DOI: 10.18609/cgti.2017.035 innovator insight addition, the temperature of ice and functionality, and is deserving nucleation, cooling rate, storage of detailed examination. However, temperature, warming rate, and fi- for the purposes of this review, the nally, the removal or dilution of the focus on biopreservation media was cryoprotectant and storage medi- chosen for further discussion as it um [20]. In addition, within a cell/ constitutes an early and critical step tissue manufacturing process, the in both hypothermic and cryogenic variability of source material qual- storage applications (Figure 2). The ity (apheresis/leukapheresis/tissue) first consideration for an optimized and the collection/transition of cells biopreservation solution is to ad- from expansion/processing to final dress the problems associated with formulation/fill are increasinglythe ionic imbalance. Rather than uti- recognized as potential bottleneck lizing an isotonic, extracellular-like stress points in the overall work- basal media composed of an ionic flow. Each of these stress points balance designed for normother- impacts post-preservation viability mic cell growth, wash or infusion,

ffFIGURE 2 Critical steps in the cryopreservation process.

Cryopreservation solution Cryoprotectant removal, choice & cryoprotectant dilution, and/or inclusion concentration in cell product

Rate of cryoprotectant addition

Temperature of cryoprotectant addition

0oC Temperature Temperature of Warming rate ice nucleation

Cooling rate

-196oC Storage temperature

Time

Even within the singular cell manufacturing step of cryopreservation, there are multiple points for Risk, and subsequently multiple points for Optimization in consideration to Biopreservation Best Practices. Modified from: Acker JP; Biopreservation and cellular therapies.ISCT Telegraft15(2), 8–11 (2008).

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cells stored at low temperatures are during freezing. In fact, the cellular recommended to be balanced in an toxicity from increased concentra- ionic composition similar to that of tion of salts is shown to be more the intracellular milieu. Doing so detrimental to cell viability than will reduce concentration gradients the CPA, in what is known to cryo- and uncontrolled ion flux across the biologists as ‘the solution effects’ cell membrane and will compen- [21]. Dimethylsulfoxide (DMSO) sate for the reduced activity of the is a membrane-permeating cryo- dysfunctional and ATP-depleted protectant that has been used for membrane pumps. Further addi- hematopoietic stem cell transplants tion of polysaccharides and other in patients for decades. And while large cell impermeant molecules early reports correlate DMSO in- increases extracellular osmolality fusion with serious adverse events, and reduces subsequent osmot- most side effects include treatable ic swelling and plasma membrane symptoms such as nausea, vomit- damage. A hypertonic solution also ing, headache and cough consistent facilitates more rapid cellular dehy- with low-grade anaphylaxis [22,23]. dration that decreases the proba- More recent studies indicate that bility of intracellular ice formation serious adverse events may not be a during freezing, and would allow result of DMSO per se, but rather greater flexibility with freezing rate. by the white blood cell/granulocyte An optimized biopreservation me- concentration of the infused prod- dia is also recommended to con- uct [24]. Cells frozen in a novel in- tain appropriate antioxidants that tracellular-like DMSO-containing minimize the impact of damaging cryopreservation solution as part free radicals generated by the mito- of a pediatric cell therapy animal chondria, as well as energetic pre- model have also been safely injected cursors to speed ATP generation directly into the hearts of neonatal upon returning to normothermia. pigs, utilizing the DMSO-contain- Optimized biopreservation media ing intracellular-like cryopreserva- are also recommended to contain tion media as an excipient without pH buffers that have a low degree of wash [25]. With a well-documented temperature sensitivity to maintain clinical history, and accepted hy- appropriate pH buffering capacity persensitivities for use in multiple during hypothermic conditions, as administration models, DMSO well as a robust capacity to stabilize continues to be the most wide- the reduction in pH associated with ly-accepted, clinically tracked, and the cellular production of lactate. effective permeating cryoprotectant The misconception among many used in cell therapy products to who deal with cryopreservation is date. For cryogenic storage, opti- that cryoprotective agents (CPAs) mized biopreservation may include are needed to minimize ice forma- permeating cryoprotectants such as tion because it is the ice that kills DMSO to minimize cellular injury the cells. While the CPA does con- due to solution effects, and it would tribute to reduced intracellular ice be recommended to also include formation, an additional way a multiple cryoprotectants that act in CPA protects cells is by minimizing both permeating and non-permeat- a significant increase in ions and ing mechanisms. As such, the po- salt concentration in the solution tential for DMSO toxicity may be

352 DOI: 10.18609/cgti.2017.035 innovator insight minimized by the concurrent use of therapy products in comparison to additional cryoprotectants that act conventional pharmaceutical and in a non-permeating mechanism. biopharmaceutical treatments [26]. Optimized cryomedia, therefore, Indeed, cell manufacturing is cur- would be designed to engineer an rently substantially more expensive effective balance of both permeat- than traditional pharmacologic and ing and non-permeating cryopro- small molecule products due to the tectants to effectively reduce the need for more complex and special- toxicity of any single cryoprotectant ized reagents, instrumentation and (such as DMSO). Alternative cryo- processes. Followed closely behind protectant options, as well as meth- cost are issues related to product ods modifications, are available to efficacy, reimbursement, safety, clinical manufacturers and patient regulations and infrastructure [26]. populations sensitive to the poten- Within all of these barriers, an im- tial clinical effects of DMSO; and portant and often overlooked ele- the risk:reward versus development ment is product stability and shelf- burden considerations are ongoing life where biopreservation comes and worthy discussions in the regen- into effect. For example, when ap- erative medicine field. Utilization proved by the FDA in 2010, Den- of an optimized biopreservation dreon’s Provenge® (sipuleucel-T) media is an early and critical step dendritic cellular immunotherapy to designing an optimized biopres- adopted a manufacturing model ervation protocol, and can poten- with short stability of the apheresis tially improve viable cell recovery starting material, and limited 18- post-preservation, and accelerate hour non-frozen shelf-life of the fin- the functional return (i.e., potency) ished product. This 18-hour prod- that is increasingly recognized as a uct stability window necessitated a critical parameter for cellular thera- complex and time-critical manufac- py clinical success. turing and supply chain with high cost-of-goods and product pricing, which has been argued was a major factor in Provenge’s limited market BIOPRESERVATION OF adoption [27]. Cell therapy compa- COMMERCIAL CELL nies have since learned the lessons THERAPY PRODUCTS of the Provenge model, and are Despite their clinical and commer- beginning to standardize product cial promise, novel cellular thera- development with an eye towards pies are nascent technologies that increased product stability at all rel- have not yet achieved widespread evant points in the needle-to-needle clinical utility and commercial via- workflow. In one noted next gener- bility. Since the US Food & Drug ation example, Kite Pharma has in- Administration (FDA) approved creased the stability of its lead oncol- Carticel as the first cellular thera- ogy product axacabtagene ciloleucel py in 1997, only a handful of ad- from 18 hours to 2 weeks by tran- ditional products have obtained sitioning to a cryopreserved prod- marketing authorizations world- uct [28], reducing manufacturing wide. The largest barrier to entry constraints and providing logistical for commercial viability involves flexibility at reduced overall cost. In the relative cost–effectiveness of cell practice, a lack of standardization

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and guidelines necessitates that systems. Recommended biopreser- each novel cell therapy developer vation media would also be free of validate biopreservation protocols animal and human serum and pro- for each product, and the varying teins to reduce the potential for dis- biopreservation methods may result ease transmission, as well as simplify in cumulative variability in efficacy, the Quality/Regulatory risk assess- costs, risks and Quality/Regulatory ment step. Elimination of serum in footprint. The standardization of particular, not only reduces the risks biopreservation media and methods and costs associated with the use of (i.e., Biopreservation Best Practices) properly vetted animal/human-de- may increase the efficiency of regen- rived products, but also facilitates erative medicine clinical develop- the GMP production by reducing ment and potential commercializa- the inherent Batch-to-Batch vari- tion, and provide a more effective ability of serum which have been roadmap for optimizing methods recognized and is almost impossible for each cell product. to characterize. The components of What constitutes Biopreservation the media and the protocol methods Best Practices for cell therapy and re- also require consideration as part of generative medicine products? With an overall Quality/Regulatory/Safety regards to an early step in the biopres- footprint, in order to appropriately ervation continuum, the selection of qualify within a GMP clinical ap- appropriate media, many non-frozen plication, and more so should the and freezing methods employ extra- biopreservation media be considered cellular-like ‘home-brew’ cocktails as an excipient within the final cell based on traditional formulations, product. The biopreservation media which often are minimally effective is recommended to have a risk pro- for research and development, but file that would allow consideration are inefficient, increase risk and are for qualification as an excipient for not scalable for GMP clinical man- direct delivery to patients, and have ufacturing. Historical home-brew clinical supporting information to cocktails may not be manufactured support the related clinical risk as- according to using USP-grade ma- sessment (Figure 3). Historical bio- terials (or multicompendial/highest preservation formulations can still quality), which may result in Batch- be compounded in-house by using to-Batch (or Lot-to-Lot) variability. traceable raw materials of the high- By contrast, Biopreservation Best est available quality (USP or mul- Practices recommendations for an ticompendial/highest quality), but optimized media would support in- would likely not be manufactured tracellular-like biopreservation me- according to GMP with appropriate dia formulation specifically designed per batch Quality Control release as- to mitigate the detrimental effects of says or stability studies, and should cold temperature storage [20,29,30]. undergo an independent internal Such intracellular-like media is rec- risk assessment for safety and stabil- ommended to be manufactured ac- ity. In lieu of in-house home-brews, cording to GMP in a certified facility commercially available alternatives (ISO, GMP, Regulatory body), with that adhere to Biopreservation Best an appropriate Quality and Regula- Practices recommendations are tory footprint and packaging options available and have been cited in a amenable to closed manufacturing number of regenerative medicine

354 DOI: 10.18609/cgti.2017.035 innovator insight

ffFIGURE 3 Parametric considerations for Biopreservation Best Practices.

Regulatory/Safety Technical parameters Donor considerations

Cell permeating/ GMP non-permeating manufacturability Source material cryoprotectant content Patient harvest Intracellular-like/ Elimination of extracellular-like serum/protein ionic balance

Well-deﬁned Storage chemistry temperature Biopreservation: Biopreservation: Transport Transport Patient logisitcs Processing logisitcs Lot-to-lot QA/QC completion Quality tests consistency

Excipient Formulation-dependent parameters use potential

Manufacturing/ Rate & temperature cell factory of addition/removal

Pre-storage incubation

Cooling/warming rate Cell-speciﬁc parameters

Selection of the right biopreservation formulation can simultaneously address multiple regulatory and technical considerations (shaded within the table on the right) and speed the path to commercialization. clinically relevant publications. Ex- and cell banks, final cell product bi- amples of such media include the opreservation and packaging, tem- intracellular-like formulations Hy- perature-controlled shipping and poThermosol FRS and CryoStor. monitoring, post-preservation pro- In addition, Biopreservation Best cessing, and patient administration. Practices recommendations would An emerging consideration within qualify methods/protocols opti- the development of regenerative mization addressing each point of medicine cell-based products is the stability risk (Figure 1), similar to a aspect of Biologistics – the processes, Failure Modes and Effects Analysis tools and data used to manage and (FMEA), throughout the cell/tissue monitor the movement of biologic lifecycle, as well as within each bio- materials across time and space. This preservation methods step (Figure 2). report does not address the aspects These methods optimization steps of Biologistics Best Practices; how- may include, but are not limited to: ever O’Donnell and colleagues have collection and transport of blood/tis- comprehensively described these sue/marrow, intermediate hold steps considerations previously [31–33].

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Cumulatively, the multiple points of quality raw materials, with an appro- potential risk and stress related to the priate Quality/Regulatory footprint workflow from collection, through that facilitates integration into cell manufacturing and supply chain lo- therapy and regenerative medicine gistics, to patient administration, are applications. Optimized biopreser- all inter-related with Biopreservation vation media would ideally be for- and Biologistics Best Practices. mulated with an intracellular-like ionic balance devoid of pathogenic animal and human proteins, and be safe for consideration as an excipient SUMMARY with appropriate qualification. In The use of manufactured cells as ‘liv- reality, the adoption of Biopreser- ing drugs’ to combat chronic disease vation Best Practices minimizes the promises to dramatically improve stresses associated with collection, human health and increase lifespan. manufacturing, storage, and trans- However, advanced cellular therapies port, improves viable and functional require specialized environmental cell recovery following biopreserva- support in order to maintain effec- tion, and ensures that cells maintain tiveness throughout the product their targeted therapeutic efficacy lifecycle. Biopreservation refers to through the product lifecycle. In do- all the practices required to mini- ing so, Biopreservation Best Practices mize cell death and damage ex vivo, reduces manufacturing costs, pro- as well as facilitate a return to func- vides logistical flexibility, and ensures tion post-preservation. Whether low therapeutic potency at the clinic. temperature biopreservation is in- Only by delivering the best possible tended for hypothermic or cryogenic treatments to patients can the true applications, there are key steps in potential of regenerative medicine the process that require optimization be fully realized, and only by man- for each cell type, including (but not ufacturing these cell/tissue products limited to): the selection of appro- to be commercially and clinically priate hypothermic/cryogenic bio- viable can these promising therapies preservation solutions and cryopro- be sustainable over the long-term to tectants, the rate of media addition, benefit the greater global population. the temperature of media addition, the temperature of ice nucleation, FINANCIAL & COMPETING cooling rate, storage temperature, INTERESTS DISCLOSURE warming rate, and the removal or BJH, AA, and AJM are employees of dilution of the storage media and BioLife Solutions, Inc. BJH and AA are cryoprotectants. Careful consider- Senior Application Scientists. AJM is ation of each of these steps can great- Senior Vice President & Chief Technology ly improve biopreservation efficacy. Officer. An early critical step in any biopres- No writing assistance was utilized in the ervation strategy is the selection of production of this manuscript. appropriate storage media, and is the primary focus of this review. Biopres- ervation Best Practices with regards This work is licensed under to media selection is recommended a Creative Commons Attri- to consider GMP-manufactured bution – NonCommercial – NoDerivatives 4.0 solutions composed of the highest International License

356 DOI: 10.18609/cgti.2017.035 innovator insight

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358 DOI: 10.18609/cgti.2017.035