Theme 5: Tissue Engineering for Regenerative Medicine

Manufacturing challenges

The auto/allo issue

Whether the product is autologous or allogeneic brings significant challenges in decision-making about the type of manufacturing model to adopt. An autologous product is one where cells are harvested from a patient and the culture expanded ex vivo to large quantities over many weeks and then returned to the patient i.e. ‘one for one’. An allogeneic product is one where culture-expanded cells originating from a single donor provide treatments to large numbers of patients i.e. ‘one for many’.

Note: Autologous products do not just include cells grown outside the body; autologous bone grafts for instance are the gold standard still for bone repair…sample of bone is taken from one part of the body [often the pelvic bone] to be used in another during one operation.

It may be that, as a general ‘rule’, clinician-instigated/innovated therapies tend to tilt towards autology while industry [read: SMEs operating like ‘small pharmas’ looking for economies of scale and a ‘warehouse’ mode of operation]-instigated/innovated therapies favour allogeneic. There are probably many exceptions to the rule, however.

Go-to persons for manufacturing

Prof David Williams, Loughborough University

Dr John Higgins, Southern Lights Biomaterials Ltd

Fig 7 presents auto/allo factors to be weighed up in making manufacturing and commercialisation decisions.

A table comparing the advantages and disadvantages of autologous and allogenic products. Advantages of autologous products are low-to-no risk of immune rejection, prevents risk of transferring pathogens from donor to host, simple procedures may be able to be automated, characterised by personalised, highly customised therapy viz one for one. The vision for autologous products is: Development of new manufacturing models for roll-out/scale-out of clinically-led autologous cell therapies. The disadvantages of autologous products are	they are difficult scale-up, May require scale-out to multiple processing facilities [distributed manufacturing], high manufacturing cost [COGs, series of QC assays for each patient, labour], Complicated logistics, Requires robust sample tracking and containment procedures, Complicated regulatory path, Cleaning validation to minimise pathogen risk during cell processing, Low quantity, low quality starting materials, Variable product quality and Possible slow adoption in clinic. The advantages of allogenic products are they may provide an ‘off-the-shelf’ product [like Pharma], controlled scale-up, Central manufacturing, with lower costs per patient [low COGs, one series of QC assays per batch], Clear logistics, Uniform and consistent product quality, Well-characterised donor cell lines, Clear and easier regulatory path
 and Potentially easy adoption in clinic. Disadvantages of allogenic products are they must overcome the risk of immune rejection [more tests required for donor raw materials and immunogenicity], Highly dependent on donor raw material availability, Shelf-life must be able to be extended to enable storage of product, Requires larger storage facility and shipment department
,Product quality and consistency must be evaluated during long-term development and commercial production, and Personalised/customised therapy not possible ex. \'one for many\'.

Fig 7 Auto/Allo factors to consider in making decisions about manufacturing and commercialisation of a CBP

The ability to avoid immunogenicity and to operate at-scale for an ‘acceptable’ cost would be an alluring proposition for many, from clinicians to small-to-medium-size enterprises [SMEs].

Autologous therapies come at a high cost per patient vis a vis allogeneic therapies but with clever thinking the economic complexion could change considerably i.e. expensive treatment but only one-off, with no immune rejection, thus lower overall cost by elimination of the need for continuing reimbursements from [ultimately] the taxpayer.

This would necessitate the development of innovative new regulatory mechanisms that recognise and accept a one-off
investment in the future health of the patient based on an officially recognised treatment of a specified medical condition.

At least half of the disadvantages in respect of autologous CBPs listed in fig 7 [specifically those coloured red] can be readily inferred by looking at the alternative routes for cell-based material travelling from donor back to herself/himself [i.e. patient].

These are shown in fig 8.

The routes constitute a multi-nodal network integrating clinical sites [biopsy, manufacturing and delivery to patient in a hospital or ‘centre of excellence’ setting], a regulated manufacturing mother site [manufacturing and supply of final product, frozen or fresh, to the clinical site], distributed manufacturing sites [possibly contract manufacturing] operationally identical to the mother site and supplying it with product, and an academic site [with both non-GMP and GMP manufacturing, and closely linked with other sites]. In practice all routes will, if the situation dictates, probably be able to be used in synchrony as one ‘parcel’ in treatment of the patient. Route selection may equally be more modest to achieve the therapeutic end result sought.

While the nodes [sites] in the network visually dominate fig 8 the importance of internodal action should not be underestimated. Road transport by courier or other delivery vehicles, for example, may have a deleterious impact on the safety and integrity of cells. Vehicle and package resonant vibrations as well as rough-road vibrations were investigated by Nikolaev et al4, who concluded that:

  • The sensitivity of human mesenchymal stem cells [hMSCs] to different vibrations within the mechanical system was similar to that observed for human dermal fibroblasts and in both cases the strongest effect was observed for a frequency of 25Hz. Vibrations at 25Hz resulted a dramatic reduction in the total cell number.
  • Long cold storage makes hMSC cells significantly susceptible to mechanical vibrations of 50Hz, peak acceleration 140m/s2, peak displacement 1.4mm.

In practice the transport conditions leading to such a result would be avoided, and conditions less unfavourable created. The work done by Nikolaev et al provides some pointers.

Overall the network offers opportunities for cost savings to ameliorate the ‘expensive treatment’ cited earlier. These opportunities include, amongst other things, modelling and reducing the cost of goods sold [COGS], commercialising new technologies that reduce cell therapy COGS, and developing and commissioning a low-cost manufacturing platform for adherent cells.

A diagram showing the alternative routes for autologous cell therapy products. Four parties are involved - Patients, Clinical Sites, Manufacturing mother site, academic site and distributed manufacturing sites.

Fig 8 Alternative routes for the manufacture, distribution and delivery of small-scale more-than-minimally-manipulated [MTMM] autologous cell therapy products5

Note: In respect of, for example, Australasia the stated transferability of product/process implies full movement within and between New Zealand and Australia on the proviso that sites are regulated according to common criteria.

Automation and comparability

Automated bioprocessing holds considerable promise for reproducible manufacturing ensuring comparability across multiple processing sites.6 This is achieved principally by eliminating manual tasks that potentially introduce high cellular variability, in part due to the exposure of cells to poorly controlled bioprocess forces. While the Regulator may call for product comparability between sites and remains detached from how this is achieved, industry is left with the challenge of, first, identifying which cellular actions are critical for the intended therapy, then, second, designing, optimising and commissioning manufacturing processes that will deliver on this as cost-effectively as possible in the same way at multiple sites.

There are differing views as to whether automation should be ‘early’ in the lab-to-manufacturing translation pathway, or ‘later’. Advocates of one strategy or the other include academics/researchers, equipment manufacturers, and financial specialists acting in the interests of their client investors. Auto/allo considerations, also, are germane, as are projected size of target markets. For autologous products targeted at small markets, especially where priority is given to first-to-market, automated processes may not necessarily be better than manual ones or even be viable.

Characterisation and control

Regardless of the type of manufacturing process, determination of instrumentation and sensing requirements for the characterisation, monitoring and control of the production of CBPs is critical. The fact that production takes place in an environment of considerable complexity [viz varying temperature, humidity, gas flow and media mix] adds to the burden. And cells' behaviour increases the complexity to be managed when researchers and their industry colleagues switch their attention from building monolayers, say up to 500μm, to tissues [or multiple tissue constructs] of more daunting 3-dimensionality.

Non-cytological factors, also, pose challenges. During the scaling process, for example, elution rates [which might amount to a few nanograms daily per injury site] of growth factors from scaffolds need to be maintained and monitored. Bioabsorption rates of scaffolds need to be kept in approximate synchrony with the growth of the tissue supported by those scaffolds.

Whether the therapeutic context is allogeneic or autologous adds to the complexity.

Specialised testing and QC approaches are required for appropriately implementing in-process and product lot-release testing for cell therapies. An array of QC tests is used for stem-cell products e.g. tests to screen for the absence of undifferentiated embryonic stem cells and tests to determine the extent of differentiation using gene expression, evaluation of cell morphology, protein production, and cell surface markers through fluorescence-activated cell sorting [FACS] analysis. Periodic testing for microbial contaminants is applied to cells or spent media for ensuring that aseptic conditions are maintained throughout processing.

Adherence to GMP

In modern/Western jurisdictions CBP manufacturing processes, again regardless of type, must be able to demonstrate adherence to GMP [Good Manufacturing Practice]. A product that conforms to GMP guidelines is considered to be of high quality and will pose no risk to consumers or the general public. As in the case of comparability the GMP guidelines do not lay out specific instructions for how to manufacturer a product; rather, they list general principles that must be observed during the manufacturing process. It is left to the individual manufacturer to decide how to set up its manufacturing system in order to best conform to the guidelines.

Logo for New Zealand Good Manufacturing Practice.

For CBPs this relates to: safety, where the risks to posed by adventitious agents, autoimmune response/immunogenicity [in allogeneic situations] and unwanted cells are negated; efficacy, where capability of the product to function in vivo [preferably in a human patient] can be demonstrated; and purity, where the performance of product when implanted in a patient can be proven by cell viability, and phenotype, function and mode of action can be shown to be consistent with therapeutic objectives.

4 The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation: N.I.Nikolaev, Y. Liu and D.J. Williams J.R. Soc. Interface published online 23 May 2012

5 Adapted by the author from Regulatory challenges for the manufacture and scale-out of autologous cell therapies: Paul.Hourd, Amit Chandra, Nick Medcalf, David J.Williams; March 2014 StemBook [Internet]

6 Comparability is the regulatory requirement to demonstrate product equivalence after a process change. Such process changes include a media component change, a donor/starting material change, a manufacturing platform change, and the introduction of a new manufacturing site.